Processes for producing isomaltose and isomaltitol and use thereof

ABSTRACT

The present invention aims to provide a novel process for producing isomaltose and isomaltitol, and uses thereof, and it solves the object by establishing a process for producing isomaltose comprising a step of contacting a saccharide, having the α-1,4 glucosidic linkage as the linkage of non-reducing end and a glucose polymerization degree of at least two, with an α-isomaltosyl-transferring enzyme and an α-isomaltosylglucosaccharide-forming enzyme derived from a specific microorganism; a process for producing isomaltitol using the isomaltose produced by the above process; saccharide compositions comprising the isomaltose and/or the isomaltitol produced by the above processes; and uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 application of PCT/JP02/10846, filedOct. 18, 2002, which claims benefit of JP 2001-321182, filed Oct. 18,2001 and JP 2002-252609, filed Aug. 30, 2002, all of which are hereinincorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a novel process for producingisomaltose and isomaltitol, and uses thereof, more particularly, to aprocess for producing isomaltose and/or isomaltitol in a relatively highyield from a saccharide which has the α-1,4 glucosidic linkage as thelinkage of non-reducing end and a glucose polymerization degree of atleast two, and uses thereof.

BACKGROUND ART

Isomaltose is a rare saccharide that merely exists in nature infermented foods in a slight amount, and it is known to be produced byconventional methods such as partial hydrolysis reactions using acidcatalysts, enzymatic reactions using dextranase or isomaltodextranase,reverse-synthetic reactions for forming isomaltose from glucose usingglucoamylase or acid catalysts, and glucose-transferring reactions forforming isomaltose from maltose or maltodextrins using α-glucosidase.However, the above conventional methods are far from a satisfactoryindustrial-scale production of isomaltose, because the isomaltosecontents in the reaction mixtures, obtained by the above conventionalprocesses, are only about 10 to about 25% (w/w) (the symbol “% (w/w)” isabbreviated as “%” throughout the specification, unless specifiedotherwise), on a dry solid basis (d.s.b.) and their purities arerelatively low. To improve this drawback, for example, a columnchromatography, disclosed in Japanese Patent Kokai No. 72,598/83, can bementioned. According to the method, a relatively high purity isomaltosecan be produced from material saccharide solutions with an isomaltosecontent of about 10 to about 25%, d.s.b. However, even if the method isemployed, there still remains a problem of that the purity and the yieldof the produced isomaltose inevitably depend on the isomaltose contentin the material saccharide solutions used.

Under these circumstances, there has been required a novel process forproducing isomaltose on an industrially scale and in a lesser cost and ahigher yield.

While isomaltitol is a sugar alcohol having satisfactorynon-reducibility, low sweetness, and moisture-retaining ability, and itis a useful sugar alcohol which has been extensively used in foodproducts, cosmetics, pharmaceuticals, etc., in the form of a saccharidemixture with sorbitol, maltitol, and glucosyl-1,6-mannitol.

Isomaltitol can be theoretically prepared by hydrogenating, i.e.,reducing the reducing group of paratinose or isomaltose, as a reducingoligosaccharide, into an alcohol group. In particular, althoughisomaltitol has been prepared from isomaltose in a relatively highyield, the desired industrial supply of material isomaltose has not beensatisfactory. Isomaltose is known to be prepared by the methods such aspartial hydrolytic reactions of dextrans using acid catalysts, enzymaticreactions using dextranase or isomaltodextranase, reverse-syntheticreaction for forming isomaltose from glucose using glucoamylase or acidcatalysts, glucose-transferring reactions for forming isomaltose frommaltose or maltodextrins using α-glucosidase. However, the aboveconventional methods are far from a satisfactory industrial-scaleproduction of isomaltitol, because the isomaltose contents in thereaction mixtures, obtained by the above conventional processes, areonly about 10 to about 25%, d.s.b., and the purity of isomaltitol,obtained by hydrogenating the above-identified isomaltose, is relativelylow. To improve the drawback, for example, by applying columnchromatography disclosed in Japanese Patent Kokai No. 72,598/83, arelatively high purity isomaltose can be obtained from materialsaccharide solutions with a relatively low isomaltose content of about10% to about 25%, d.s.b., and then hydrogenated to obtain isomaltitol.Even in the process for producing isomaltitol, as a drawback, the yieldand the cost of isomaltitol inevitably depend on the isomaltose contentof the material saccharide solutions used, and this lowers the yield andincreases the production cost of isomaltitol.

While in the case of producing isomaltitol from paratinose, the materialparatinose is known to be prepared, for example, from sucrose throughglucose-transferring reaction using α-glucosyl transferase. However,since the resulting reaction mixture comprises, as by products,trehalulose as an isomer of paratinose and others such as glucose andfructose as hydrolyzates of paratinose, the paratinose content in thereaction mixture could not be over about 85%, d.s.b. In producingisomaltitol from paratinose, glucosyl-1,6-mannitol is formed along withisomaltitol in a production ratio of, usually, 1:1 by weight, and thislowers the purity and the yield of isomaltitol as a drawback.

Under these circumstances, a novel process for producing isomaltitol onan industrial scale and in a lesser cost and a higher yield has beenstrongly required.

DISCLOSURE OF INVENTION

Considering the above prior arts, the object of the present invention isto establish a process for producing isomaltose and isomaltitol on anindustrial scale and in a lesser cost and a higher yield, and usesthereof. Namely, the object of the present invention is to establish aprocess for producing isomaltose and isomaltitol on an industrial scaleand in a lesser cost and a higher yield, saccharide mixtures comprisingisomaltose and/or isomaltitol, and uses thereof.

During the present inventors had been eagerly studying on solving theabove objects, it was reported in European Journal of Biochemistry, Vol.226, pp. 641-648 (1994) a cyclic tetrasaccharide, having the structureofcyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→}(may be called “cyclotetrasaccharide” throughout the specification),having the structure of isomaltose intramolecularly, formed bycontacting a hydrolyzing enzyme, i.e., alternanase, with alternancomposed of four glucose molecules linked together via the alternatingα-1,3 and α-1,6 bonds.

As previously disclosed in Japanese Patent Application No. 2000-229557(International Publication No. WO 01/90338), the present inventorsestablished a process for producing cyclotetrasaccharide using anα-isomaltosyl-transferring enzyme which forms cyclotetrasaccharide fromamylaceous saccharides such as panose, and in Japanese PatentApplication No. 2000-234937 (International Publication No. WO 02/10361),they established another process for producing cyclotetrasaccharide in ahigher yield by allowing an α-isomaltosyl-transferring enzyme and anα-isomaltosylglucosaccharide-forming enzyme which formsα-isomaltosylglucosaccharide from maltooligosaccharides. Further, asdisclosed in Japanese Patent Application No. 2001-130922 (InternationalPublication No. WO 02/04166), the present inventors established anotherprocess for producing isomaltose in a higher yield by allowing anα-isomaltosylglucosaccharide-forming enzyme and an isomaltose-releasingenzyme to act on material starches.

Thereafter, the present inventors discoveredα-isomaltosylglucosaccharides and anα-isomaltosylglucosaccharide-forming enzyme, which can be used in theabove process for producing isomaltose, and also found that isomaltoseis produced on an industrial scale and in a lesser cost and a higheryield by using these enzymes. The present inventors further studied themethod for producing isomaltitol from isomaltose; they studied theenzymatic reaction mechanisms of suchα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme and found that the production yield ofisomaltose is dramatically increased by allowing anα-isomaltosylglucosaccharide-forming enzyme and anα-isomaltose-releasing enzyme capable of releasing isomaltose to act ona saccharide having the α-1,4 glucosidic linkage as the linkage ofnon-reducing end and a glucose polymerization degree of at least two inthe presence or the absence of α-isomaltosyl-transferring enzyme, andthat isomaltitol is easily produced on an industrial scale and in anincreased yield by hydrogenating the isomaltose thus obtained. Thepresent inventors also established the uses of the isomaltitol thusobtained and accomplished this invention; they solved the above objectby establishing a process for producing isomaltose comprising a step ofcontacting a saccharide, having the α-1,4 glucosidic linkage as thelinkage of non-reducing end and a glucose polymerization degree of atleast two, with one or more α-isomaltosylglucosaccharide-forming enzymesderived from Bacillus globisporus N75 strain (FERM BP-7591) (hereinaftermay be called “N75 strain”), Arthrobacter globiformis A19 strain (FERMBP-7590) (hereinafter may be called “A19 strain”), and Arthrobacterramosus S1 strain (FERM BP-7592) (hereinafter may be called “S1strain”), which are disclosed in PCT/JP01/06412 (InternationalPublication No. WO 02/10361) in the presence or the absence ofα-isomaltosyl-transferring enzyme derived from Bacillus globisporus N75strain (FERM BP-7591) and/or Arthrobacter globiformis A19 strain (FERMBP-7590) to form α-isomaltosylglucosaccharides having the α-1,6glucosidic linkage as the linkage of non-reducing end and the α-1,4glucosidic linkage other than the above linkage, and/or to form asaccharide with the structure ofcyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→},contacting the products thus obtained with isomaltose-releasing enzymeto form isomaltose, and collecting the produced isomaltose; saccharidemixtures with such isomaltose; and uses thereof. As regards theabove-identified Bacillus globisporus N75 strain (FERM BP-7591), themicroorganism was deposited on May 16, 2001, and has been maintained inInternational Patent Organism Depositary National Institute of AdvancedIndustrial Science and Technology, AIST Tsukuba Central 6, 1-1, Higashi1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan. Arthrobacter ramosusS1 strain (FERM BP-7592) was deposited on May 16, 2001, and has beenmaintained in the above institute.

The present inventors further solved the object of the present inventionby contacting a saccharide, having the α-1,4 glucosidic linkage as thelinkage of non-reducing end and a glucose polymerization degree of atleast two, with α-isomaltosylglucosaccharide-forming enzyme in thepresence or the absence of α-isomaltosyl-transferring enzyme to formα-isomaltosylglucosaccharides, having the α-1,6 glucosidic linkage asthe linkage of non-reducing end and α-1,4 glucosidic linkage other thanthe above linkage and having a glucose polymerization degree of at leastthree, and/or cyclotetrasaccharide; contacting the resulting saccharideswith isomaltose-releasing enzyme to form isomaltose; hydrogenating theresulting mixtures containing isomaltose directly or after collectingisomaltose to form isomaltitol; and collecting the formed isomaltitol;saccharide mixtures containing isomaltitol; and uses thereof.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an elution pattern of a saccharide, obtained by the enzymaticreaction using α-isomaltosyl-transferring enzyme from a microorganism ofthe species Bacillus globisporus C9 strain, when determined onhigh-performance liquid chromatography.

FIG. 2 is a spectrum of nuclear magnetic resonance (¹H-NMR) ofcyclotetrasaccharide, obtained by the enzymatic reaction usingα-isomaltosyl-transferring enzyme from a microorganism of the speciesBacillus globisporus C9 strain.

FIG. 3 is a spectrum of nuclear magnetic resonance (¹³C-NMR) ofcyclotetrasaccharide, obtained by the enzymatic reaction usingα-isomaltosyl-transferring enzyme from a microorganism of the speciesBacillus globisporus C9 strain.

FIG. 4 represents the structure of cyclotetrasaccharide, i.e.,cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→}.

FIG. 5 shows the thermal influence on the enzymatic activity ofα-isomaltosylglucosaccharide forming enzyme from a microorganism of thespecies Bacillus globisporus C9 strain.

FIG. 6 shows the pH influence on the enzymatic activity ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Bacillus globisporus C9 strain.

FIG. 7 shows the thermal stability ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Bacillus globisporus C9 strain.

FIG. 8 shows the pH stability of α-isomaltosylglucosaccharide-formingenzyme from a microorganism of the species Bacillus globisporus C9strain.

FIG. 9 shows the thermal influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesBacillus globisporus C9 strain.

FIG. 10 shows the pH influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesBacillus globisporus C9 strain.

FIG. 11 shows the thermal stability of α-isomaltosyl-transferring enzymefrom a microorganism of the species Bacillus globisporus C9 strain.

FIG. 12 shows the pH stability of α-isomaltosyl-transferring enzyme froma microorganism of the species Bacillus globisporus C9 strain.

FIG. 13 shows the thermal influence on the enzymatic activity ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Bacillus globisporus C11 strain.

FIG. 14 shows the pH influence on α-isomaltosylglucosaccharide-formingenzyme from a microorganism of the species Bacillus globisporus C11strain.

FIG. 15 shows the thermal stability ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Bacillus globisporus C11 strain.

FIG. 16 shows the pH stability of α-isomaltosylglucosaccharide-formingenzyme from a microorganism of the species Bacillus globisporus C11strain.

FIG. 17 shows the thermal influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesBacillus globisporus C11 strain.

FIG. 18 shows the pH influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesBacillus globisporus C11 strain.

FIG. 19 shows the thermal stability of α-isomaltosyl-transferring enzymefrom a microorganism of the species Bacillus globisporus C11 strain.

FIG. 20 shows the pH stability of α-isomaltosyl-transferring enzyme froma microorganism of the species Bacillus globisporus C11 strain.

FIG. 21 shows the thermal influence on the enzymatic activity ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Bacillus globisporus N75 strain.

FIG. 22 shows the pH influence on the enzymatic activity ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Bacillus globisporus N75 strain.

FIG. 23 shows the thermal stability ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Bacillus globisporus N75 strain.

FIG. 24 shows the pH stability of α-isomaltosylglucosaccharide-formingenzyme from a microorganism of the species Bacillus globisporus N75strain.

FIG. 25 shows the thermal influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesBacillus globisporus N75 strain.

FIG. 26 shows the pH influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesBacillus globisporus N75 strain.

FIG. 27 shows the thermal stability of α-isomaltosyl-transferring enzymefrom a microorganism of the species Bacillus globisporus N75 strain.

FIG. 28 shows the pH stability of α-isomaltosyl-transferring enzyme froma microorganism of the species Bacillus globisporus N75 strain.

FIG. 29 shows the thermal influence on the enzymatic activity ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Arthrobacter globiformis A19 strain.

FIG. 30 shows the pH influence on the enzymatic activity ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Arthrobacter globiformis A19 strain.

FIG. 31 shows the thermal stability ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism of thespecies Arthrobacter globiformis A19 strain.

FIG. 32 shows the pH stability of α-isomaltosylglucosaccharide-formingenzyme from a microorganism of the species Arthrobacter globiformis A19strain.

FIG. 33 shows the thermal influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesArthrobacter globiformis A19 strain.

FIG. 34 shows the pH influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesArthrobacter globiformis A19 strain.

FIG. 35 shows the thermal stability of α-isomaltosyl-transferring enzymefrom a microorganism of the species Arthrobacter globiformis A19 strain.

FIG. 36 shows the pH stability of α-isomaltosyl-transferring enzyme froma microorganism of the species Arthrobacter globiformis A19 strain.

FIG. 37 is a figure for a restriction map of a recombinant DNA “pAGA4”,where the part with a bold line is a DNA encoding a polypeptide havingan α-isomaltosyl-transferring enzyme activity, derived from amicroorganism of the species Arthrobacter globiformis A19 strain.

FIG. 38 shows the thermal influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesArthrobacter ramosus S1 strain.

FIG. 39 shows the pH influence on the enzymatic activity ofα-isomaltosyl-transferring enzyme from a microorganism of the speciesArthrobacter ramosus S1 strain.

FIG. 40 shows the thermal stability of α-isomaltosyl-transferring enzymefrom a microorganism of the species Arthrobacter ramosus S1 strain.

FIG. 41 shows the pH stability of α-isomaltosyl-transferring enzyme froma microorganism of the species Arthrobacter ramosus S1 strain.

FIG. 42 is a spectrum of nuclear magnetic resonance (¹H-NMR) ofα-isomaltosylmaltotriose, obtained by the enzymatic reaction usingα-isomaltosylglucosaccharide-forming enzyme.

FIG. 43 is a spectrum of nuclear magnetic resonance (¹H-NMR) ofα-isomaltosylmaltotetraose, obtained by the enzymatic reaction usingα-isomaltosylglucosaccharide-forming enzyme.

FIG. 44 is a spectrum of nuclear magnetic resonance (¹³C-NMR) ofα-isomaltosylmaltotriose, obtained by the enzymatic reaction usingα-isomaltosylglucosaccharide-forming enzyme.

FIG. 45 is a spectrum of nuclear magnetic resonance (¹³C-NMR) ofα-isomaltosylmaltotetraose, obtained by the enzymatic reaction usingα-isomaltosylglucosaccharide-forming enzyme.

FIG. 46 is a spectrum of nuclear magnetic resonance (¹H-NMR) of productA.

FIG. 47 is a spectrum of nuclear magnetic resonance (¹³C-NMR) of productA.

FIG. 48 is an x-ray powder diffraction pattern of isomaltitol crystalobtained by the method of the present invention.

FIG. 49 is a spectrum of nuclear magnetic resonance (¹H-NMR) ofisomaltitol crystal obtained by the method of the present invention.

FIG. 50 is a spectrum of nuclear magnetic resonance (¹³C-NMR) ofisomaltitol crystal obtained by the method of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The α-isomaltosylglucosaccharide-forming enzyme as referred to as in thepresent invention means those which forms α-isomaltosylglucosaccharidessuch as α-isomaltosylglucose (or panose), α-isomaltosylmaltose,α-isomaltosylmaltotriose, and α-isomaltosyltetraose;α-isomaltosylglucosaccharide-forming enzymes derived from microorganismsof the species Bacillus globisporus C9 strain (FERM BP-7143)(hereinafter may be called “C9 strain”), Bacillus globisporus C11 strain(FERM BP-7144) (hereinafter may be called “C11 strain”), Bacillusglobisporus N75 strain (FERM BP-7591), and Arthrobacter globiformis A19strain (FERM BP-7590), which are disclosed in PCT/JP01/06412(International Publication No. WO 02/10361); and recombinantpolypeptides having an activity of α-isomaltosylglucosaccharide-formingenzyme, which is disclosed in Japanese Patent Application No. 2001-5441(International Publication No. WO 02/055708). Among these enzymes, thosefrom Bacillus globisporus N75 strain (FERM BP-7591) and Arthrobacterglobiformis A19 strain (FERM BP-7590) are most preferably used in thepresent invention. As regards the above-identified Bacillus globisporusC9 strain (FERM BP-7143), and Bacillus globisporus C11 strain (FERMBP-7144) were deposited on Apr. 25, 2000, and have been maintained inNational Institute of Bioscience and Human-Technology Agency ofIndustrial Science and Technology, now changed into International PatentOrganism Depositary National Institute of Advanced Industrial Scienceand Technology, AIST Tsukuba Central 6, 1-1, Higashi 1-Chome,Tsukuba-shi, Ibaraki-ken, 305-8566, Japan.

The α-isomaltosylglucosaccharide-forming enzyme as referred to as in thepresent invention is a generic term for enzymes and polypeptides whichhave an activity of α-isomaltosylglucosaccharide-forming enzyme, and itis an enzyme which forms, via the α-glucosyl-transfer, a saccharide,having a glucose polymerization degree of at least three and having boththe α-1,6 glucosidic linkage as the linkage of non-reducing end and theα-1,4 glucosidic linkage other than the above linkage, from a materialsaccharide having a glucose polymerization degree of at least two andhaving the α-1,4 glucosidic linkage as the linkage of non-reducing end,without substantially increasing the reducing power of the materialsaccharide used; has no dextran-forming ability; and which is inhibitedby EDTA (ethylenediaminetetraacetic acid). More particularly, the abovematerial saccharide, having both a glucose polymerization degree of atleast two and the α-1,4 glucosidic linkage as the linkage ofnon-reducing end, includes, for example, one or more saccharidesselected from maltooligosaccharides, maltodextrins, amylodextrins,amyloses, amylopectins, soluble starches, gelatinized starches, andglycogens. The above α-isomaltosylglucosaccharide-forming enzyme has thefollowing physicochemical properties:

(1) Action

-   -   Forming a saccharide having a glucose polymerization degree of        at least three and having both the α-1,6 glucosidic linkage as        the linkage at the non-reducing end and the α-1,4 glucosidic        linkage other than the above linkage, via the        α-glucosyl-transfer from a material saccharide having a glucose        polymerization degree of at least two and having the α-1,4        glucosidic linkage as the linkage at the non-reducing end,        without substantially increasing the reducing power of the        material saccharide;

(2) Molecular weight

-   -   Having a molecular weight of about 74,000 to about 160,000        daltons when determined on SDS-PAGE (sodium dodecyl sulfate        polyacrylamide gel electrophoresis);

(3) Isoelectric point

-   -   Having an isoelectric point of about 3.8 to about 7.8 when        determined on isoelectrophoresis using ampholine;

(4) Optimum temperature

-   -   Having an optimum temperature of about 40° C. to about 50° C.        when incubated at a pH of 6.0 for 60 min;    -   Having an optimum temperature of about 45° C. to about 55° C.        when incubated at a pH of 6.0 for 60 min in the presence of 1 mM        Ca²⁺;    -   Having an optimum temperature of 60° C. when incubated at a pH        of 8.4 for 60 min; or    -   Having an optimum temperature of 65° C. when incubated at a pH        of 8.4 for 60 min in the presence of 1 mM Ca²⁺;

(5) Optimum pH

-   -   Having an optimum pH of about 6.0 to about 8.4 when incubated at        35° C. for 60 min;

(6) Thermal stability

-   -   Having a thermostable region at temperatures of about 45° C. or        lower when incubated at a pH of 6.0 for 60 min,    -   Having a thermostable region at temperatures of about 50° C. or        lower when incubated at a pH of 6.0 for 60 min in the presence        of 1 mM Ca²⁺,    -   Having a thermostable region at temperatures of about 55° C. or        lower when incubated at a pH of 8.0 for 60 min, and    -   Having a thermostable region at temperatures of about 60° C. or        lower when incubated at a pH of 8.0 for 60 min in the presence        of 1 mM Ca²⁺;

(7) pH Stability

-   -   Having a stable pH region at about 4.5 to about 10.0 when        incubated at 4° C. for 24 hours; and

(8) N-Terminal amino acid sequence

-   -   tyrosine-valine-serine-serine-leucine-glycine-asparagine-leucine-isoleucine,        histidine-valine-serine-alanine-leucine-glycine-asparagine-leucine-leucine,        alanine-proline-leucine-glycine-valine-glutamine-arginine-alanine-glutamine-phenylalanine-glutamine-serine-glycine,        or others.

The α-isomaltosyl-transferring enzyme used in the present inventionmeans an enzyme, which forms cyclotetrasaccharide fromα-isomaltosylglucosaccharides such as panose and isomaltosylmaltose, forexample, α-isomaltosyl-transferring enzymes derived from Bacillusglobisporus C9 strain (FERM BP-7143), Bacillus globisporus C11 strain(FERM BP-7144), Bacillus globisporus N75 strain (FERM BP-7591),Arthrobacter globiformis A19 strain (FERM BP-7590), and Arthrobacterramosus S1 strain (FERM BP-7592), as well as recombinant polypeptideshaving an activity of α-isomaltosyl-transferring enzyme disclosed inPCT/JP01/10044 (International Publication No. WO 02/40659), which allhave an α-isomaltosyl-transferring activity and are called as a generalterm of “α-isomaltosyl-transferring enzyme” in the present invention.Among these enzymes, those from Bacillus globisporus N75 strain (FERMBP-7591), Arthrobacter globiformis A19 strain (FERM BP-7590), andArthrobacter ramosus S1 strain (FERM BP-7592) are most preferably usedin the present invention. The α-isomaltosyl-transferring enzyme usablein the present invention has the following physicochemical properties:

(1) Action

-   -   Forming a cyclotetrasaccharide having the structure of        cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→}        from a saccharide having a glucose polymerization degree of at        least three and having both the α-1,6 glucosidic linkage as the        linkage at the non-reducing end and the α-1,4 glucosidic linkage        other than the above linkage;

(2) Molecular weight

-   -   Having a molecular weight of about 82,000 to about 136,000        daltons when determined on SDS-PAGE;

(3) Isoelectric point (pI)

-   -   Having a pI of about 3.7 to about 8.3 when determined on        isoelectrophoresis using ampholine;

(4) Optimum temperature

-   -   Having an optimum temperature of about 45° C. to about 50° C.        when incubated at a pH of 6.0 for 30 min;

(5) Optimum pH

-   -   Having an optimum pH of about 5.5 to about 6.5 when incubated at        35° C. for 30 min;

(6) Thermal stability

-   -   Having a thermostable range at temperatures of about 45° C. or        lower when incubated at a pH of 6.0 for 60 min;

(7) pH Stability

-   -   Having a stable pH range at about 3.6 to about 10.0 when        incubated at 4° C. for 24 hours.

(8) N-Terminal amino acid sequence

-   -   isoleucine-aspartic        acid-glycine-valine-tyrosine-histidine-alanine-proline,    -   aspartic        acid-threonine-leucine-serine-glycine-valine-phenylalanine-histidine-glycine-proline,        or others.

The isomaltose-releasing enzyme used in the present invention means anenzyme, which has an action of releasing isomaltose fromα-isomaltosylglucosaccharides or cyclotetrasaccharide, such asisomaltodextranase (EC 3.2.1.94) derived from microorganisms of thespecies Arthrobacter globiformis T6 (NRRL B-4425) reported in Journal ofBiochemistry, Vol. 75, pp. 105-112 (1974); Arthrobacter globiformis (IAM12103) which is distributed and available from Institute of Molecularand Cellular Biosciences, the University of Tokyo, Tokyo, Japan; andActinomadura R10 (NRRL B-11411) disclosed in Carbohydrate Research, Vol.89, pp. 289-299 (1981).

The saccharide usable in the present invention, which has the α-1,4glucosidic linkage as the linkage of non-reducing end and a glucosepolymerization degree of at least two, means one or more saccharidesselected from maltooligosaccharides, maltodextrins, amylodextrins,amyloses, amylopectins, soluble starches, liquefied starches,gelatinized starches, and glycogens. Examples of material starches forthe above-identified soluble starches, liquefied starches, andgelatinized starches are, for example, terrestrial starches such ascorns, rices, and wheats; subterranean starches such as potatoes, sweetpotatoes, and tapioca; and partial hydrolyzates thereof, i.e., partialstarch hydrolyzates. Preferably, such partial starch hydrolyzates can begenerally prepared by suspending the above terrestrial or subterraneanstarches in water into starch suspensions with a concentration, usually,of at least 10%, preferably, 15 to 65%, and more preferably, 20 to 50%;and liquefying the starch suspensions with acids or enzyme preparations.The liquefaction degree of the above terrestrial and subterraneanstarches is preferably set to a relatively low level, usually, a DE(dextrose equivalent) of less than 15, preferably, a DE of less than 10,and more preferably, DE of 9 to 0.1. In the case of liquefying the aboveterrestrial or subterranean starches with acids, for example, employedare methods which comprise the steps of liquefying the starches withacids such as hydrochloric acid, phosphoric acid, and oxalic acid; andthen usually neutralizing the resulting mixtures with one or morealkalis such as calcium carbonate, calcium oxide, and sodium carbonateto adjust the mixtures to a desired pH. In the case of liquefying theabove terrestrial or subterranean starches with an enzyme such asα-amylase, particularly, thermostable liquefying α-amylase can bepreferably used as such an enzyme in the present invention. Isomaltosecan be obtained in a higher yield by contacting saccharides, having theα-1,4 glucosidic linkage as the linkage of their non-reducing ends and aglucose polymerization degree of at least two, withα-isomaltosylglucosaccharide-forming enzyme in the presence or theabsence of α-isomaltosyl-transferring enzyme to formcyclotetrasaccharide and/or α-isomaltosylglucosaccharides having theα-1,6 glucosidic linkage as the linkage of their non-reducing ends andthe α-1,4 glucosidic linkage as a linkage other than that of theirnon-reducing ends; and contacting the formed saccharides withisomaltose-releasing enzyme to form isomaltose; and collecting theformed isomaltose. In the case of contacting the terrestrial orsubterranean starches with α-isomaltosylglucosaccharide-forming enzymein the presence or the absence of α-isomaltosyl-transferring enzyme, oneor more enzymes selected from α-isomaltosyl-transferring enzyme,cyclomaltodextrin glucanotransferase (abbreviated as “CGTase”hereinafter), α-glucosidase, glucoamylase, and starch debranching enzymeincluding isoamylase and pullulanase can be used in combination; or oneor more enzymes selected from α-isomaltosyl-transferring enzyme, CGTase,α-glucosidase, glucoamylase, and isoamylase can be used after the actionof α-isomaltosylglucosaccharide-forming enzyme in the presence or theabsence of α-isomaltosyl-transferring enzyme, whereby isomaltose can beformed in a relatively high yield. In particular, the production yieldof isomaltose from cyclotetrasaccharide can be increased to 100% as thehighest possible level by allowing isomaltose-releasing enzyme to act oncyclotetrasaccharide, prepared by contactingα-isomaltosylglucosaccharide-forming enzyme with saccharides, havingα-1,4 glucosidic linkage as the linkage of non-reducing end and aglucose polymerization degree of at least two, in the presence ofα-isomaltosyl-transferring enzyme. In practicing the present invention,the order of the enzymes used can be decided depending on the desiredproduction yield of isomaltose, reaction time, reaction condition, etc.,a plurality of enzymes can be used simultaneously; or a requisite amountof enzymes can be divided into portions and used at different timings.The pH for the enzymatic reactions of the enzymes used in the presentinvention is usually in the range of pH 4 to 10, preferably, pH 5 to 9.The temperature for the enzymatic reactions of the enzymes used in thepresent invention is usually in the range of 10 to 80° C., preferably,30 to 70° C. The amount of enzymes used can be appropriately setdepending on the reaction conditions and reaction times for each enzyme,and it is usually appropriately selected from 0.01 to 100 units/gsubstrate for α-isomaltosyl-transferring enzyme andα-isomaltosylglucosaccharide-forming enzyme, 1 to 10,000 units/gsubstrate for isomaltose-releasing enzyme and starch debranching enzyme,and 0.05 to 7,000 units/g substrate for CGTase, α-glucosidase,glucoamylase, and isoamylase. Varying depending on the amount of theenzymes used, the reaction time is appropriately set in view of theaimed production yield of isomaltose, usually, it is set to terminatethe whole enzymatic reactions within 1 to 200 hours, preferably, 5 to150 hours, and more preferably, 10 to 100 hours. The pH and temperatureduring each enzymatic reaction can be appropriately altered beforecompletion of the enzymatic reactions of the present invention.

The content of isomaltose in the enzymatic reaction mixtures thusobtained usually reaches at least 30%, preferably, at least 40%, morepreferably, at least 50%, and more preferably, 99% or more as thehighest possible level. Particularly, enzymatic reaction mixtures havingan isomaltose content of at least 50%, d.s.b., can be easily obtained bycontacting α-isomaltosylglucosaccharide-forming enzyme,α-isomaltosyl-transferring enzyme, and isomaltose-releasing enzymesimultaneously or in this order with saccharides having the α-1,4glucosidic linkage as the linkage of non-reducing end and a glucosepolymerization degree of at least two. The above enzymatic reactionmixtures are usually subjected to conventional methods of filtration andcentrifugation to remove insoluble impurities, followed by desalting topurify the resulting mixtures with ion exchangers in H- and OH-forms,and concentrating the resultants into syrups. The resulting syrups canbe dried into solid or powdery products. If necessary, the above syrupsand products can be purified into high isomaltose content products byusing one or more fractionations using column chromatography usingion-exchangers, activated charcoals, and silica gels, etc.; separationsusing organic solvents such as alcohols and acetone; and separationmethods using membranes, which can be used in an appropriatecombination. In particular, as an industrial scale production method forhigh isomaltose content products, column chromatography usingion-exchange resins is advantageously used; column chromatography usingone or more strong-acid cation exchange resins in an alkaline metal formof Na⁺, etc., or alkaline earth metal forms of Ca²⁺, Mg²⁺, etc., ofstyrene-divinylbenzene cross-linked copolymer resins with sulfonicgroup, as disclosed, for example, in Japanese Patent Kokai Nos.23,799/83 and 72,598/83, facilitates the production of high isomaltosecontent products on an industrial scale and in a relatively high yieldand low cost. Examples of commercialized products of theabove-identified strong-acid cation exchange resins are “DOWEX 50W-X2™”,“DOWEX 50W-X4™”, and “DOWEX 50W-X8™”, commercialized by Dow ChemicalCo., Midland, Mich., USA; “AMBERLITE CG-120™” commercialized by Rohm &Hass Company, PA, USA; “XT-1022E™”, commercialized by Tokyo OrganicChemical Industries, Ltd., Tokyo, Japan; “DIAION SK1B™”, “DIAIONSK102™”, “DIAION SK104™”, etc., which are cation exchangerscommercialized by Mitsubishi Chemical Corporation, Tokyo, Japan. Inpracticing such column chromatography using the above ion-exchangeresins, any one of fixed-bed, moving bed, and semi-moving methods can beemployed. With these methods, isomaltose can be increased its purity,d.s.b., usually, up to 60% or more, preferably, 80% or more, and morepreferably, 99% or more, as the highest possible purity, in a relativelyhigh yield. High isomaltose content products other than the isomaltosewith the highest possible purity usually comprise isomaltose and one ormore saccharides selected from glucose, maltose, maltotriose,maltotetraose, other partial starch hydrolyzates,α-isomaltosylglucosaccharide, cyclotetrasaccharide, andα-glucosyl-(1→6)-α-glucosyl-(1→3)-α-glucosyl-(1→6)-glucose (hereinaftermay be abbreviated as “ring-opened tetrasaccharide”) in a total amount,excluding that of isomaltose, usually, of 1 to 60%, d.s.b. Toindustrially produce isomaltitol by hydrogenating isomaltose, theabove-identified desalting and purification steps using ion-exchangersin H- and OH-forms can be omitted, if necessary.

By hydrogenating the resulting isomaltose or isomaltose-containingproducts in the presence of reducing catalysts, isomaltitol and highisomaltitol content products can be produced in a relatively high yield.For example, the Raney Nickel catalyst is added to a 40-60% aqueousisomaltose solution. The mixture is placed in a high-pressure vessel,filled with hydrogen, increased its inner pressure, and stirred attemperatures of 100 to 120° C. to hydrogenate the isomaltose until thehydrogen is no more consumed. In this case, isomaltose is reduced toisomaltitol, while reducing saccharides contained inisomaltose-containing products, such as glucose, maltose, maltotetraose,other partial starch hydrolyzates, reducingα-isomaltosylglucosaccharide, and ring-opened tetrasaccharide aresimultaneously reduced to sugar alcohols. Cyclotetrasaccharide is anon-reducing saccharide which is not susceptible to hydrogenation. Afterremoving the Raney nickel catalyst from the resulting isomaltitolsolution, the resulting solution is decolored with activated charcoal,desalted for purification with ion-exchangers in H- and OH-forms, andconcentrated into a syrupy product, and optionally further dried into apowdery product. In necessary, the syrupy product can be, for example,purified by one or more of the following methods alone or in anappropriate combination into a saccharide mixture with isomaltitol:Fractionation of column chromatography using ion-exchangers, activatedcharcoals, silica gels, etc.; crystallization; separation using organicsolvents such as alcohols and acetone; and separation using membranes.The crystallization method for isomaltitol is usually effected byplacing in a crystallizer a supersaturated solution of isomaltitol keptat 40 to 95° C., gradually adding a seed to the solution in an amount,usually, of 0.1 to 20%, and gradually cooling the mixture under gentlystirring conditions to crystallize the contents and to form amassecuite. Thereafter, the resulting massecuite is subjected toconventional methods such as separation, block pulverization,fluidized-bed granulation, and spray drying to obtain a powderycrystalline isomaltitol, which is usually an anhydrous crystallineisomaltitol. The above separation means usually a method for separatingmassecuite into isomaltitol crystal and syrup by using a basket-typecentrifuge, where a small amount of cooled water is optionally sprayedover the formed crystal for washing to facilitate the production ofnon-hygroscopic crystalline isomaltitol with a higher purity. As regardsthe other three methods among the above-identified methods, they have acharacteristic of a higher yield of crystalline isomaltitol, althoughthe purity of isomaltitol in the resulting massecuite with crystallineisomaltitol is not substantially improved because they do not separatesyrup. Therefore, such massecuite usually comprises crystallineisomaltitol and one or more saccharides from sorbitol, maltitol,maltotriitol, maltotetraitol, sugar alcohols derived from other partialstarch hydrolyzates and α-isomaltosylglucosaccharides,cyclotetrasaccharide, andα-glucosyl-(1→6)-α-glucosyl-(1→3)-α-glucosyl-(1→6)-sorbitol (hereinaftermay be abbreviated as “reduced ring-opened tetrasaccharide”). In thecase of spray drying, a massecuite with a concentration of 70 to 85% anda crystallization percentage of 25 to 60 is sprayed from a nozzle by ahigh-pressure pump; dried with air heated to a temperature, free ofmelting the formed powdery crystal, usually, a temperature of 60 to 100°C.; and aged by blowing air heated to 30 to 60° C. for about 1 to about20 hours to facilitate the production of a non-hygroscopic orsubstantially-hygroscopic crystal with syrup. In the case of blockpulverization, usually, a massecuite with a concentration of 85 to 95%and a crystallization percentage of about 10 to about 60% are allowed tostand for 0.5 to 5 days to crystallize and solidify the whole contentsinto a block, followed by pulverizing the block by the methods such ascrushing and cutting, and drying the resultant to facilitate theproduction of a non-hygroscopic or substantially-hygroscopic crystalwith syrup.

With these crystallization methods, isomaltitol with a purity, usually,of at least 40%, d.s.b., preferably, at least 60%, d.s.b., and morepreferably, at least 99%, d.s.b., can be obtained in a higher yield.Also, saccharide mixtures with isomaltitol, which comprise maltitol andone or more saccharides from sorbitol, maltitol, maltotriitol,maltotetraitol, sugar alcohols prepared from other partial starchhydrolyzates and α-isomaltosylglucosaccharides, cyclotetrasaccharide,and reduced ring-opened tetrasaccharide in a total amount excluding thatof isomaltitol, usually, of not higher than 70%, d.s.b., preferably, nothigher than 60%, d.s.b., and more preferably, 1 to 50%, d.s.b., can beeasily obtained. Among the aforementioned saccharide mixtures withisomaltitol, those, which comprise isomaltitol and one or moresaccharides from cyclotetrasaccharide, reduced ring-openedtetrasaccharide, sorbitol, maltitol, maltotriitol, maltotetraitol, andsugar alcohols prepared from other partial starch hydrolyzates andα-isomaltosylglucosaccharide, are novel compositions. Examples of theform of the isomaltitol and saccharide mixtures with isomaltitolobtained by the present process include various forms of liquids,pastes, syrups, granules, powders, and solids.

The isomaltose, isomaltitol, saccharide mixtures of isomaltose and/orisomaltitol, and crystalline isomaltitol (which all may be generallycalled “the saccharides of the present invention” hereinafter) producedby the process of the present invention have a high quality and elegantsweetness, and have a feature of that they do not substantially formacids, as a causative of dental caries, by dental caries-inducingmicroorganisms. Thus, the saccharides of the present invention can bepreferably used as sweeteners which do not substantially induce dentalcaries. Varying to some extent depending on the purity of isomaltose andisomaltitol, the saccharides of the present invention have substantiallynon- or insubstantial-hygroscopicity, satisfactory free-flowing ability,and desired shelf-life, do not substantially induce the Maillardreaction even in the presence of amino compounds such as amino acids andproteins, do not substantially affect the coexisting ingredients, and donot substantially change color in themselves. The saccharide mixturesand products with crystalline isomaltitol according to the presentinvention can be advantageously used as a sugar coating for tablet incombination with one or more conventional binders such as pullulan,hydroxyethyl starch, and polyvinylpyrrolidone. The saccharides of thepresent invention have also useful properties of osmosis-controllingability, filler-imparting ability, gloss-imparting ability, ability ofsaccharide-crystallization-preventing ability, substantialnon-fermentability, and starch-retrogradation-preventing ability. Thus,the saccharides according to the present invention can be arbitrarilyused as a sweetener, taste-improving agent, flavor-improving agent,flavor-retaining agent, quality-improving agent, stabilizer,filler-imparting agent in various compositions such as food productsincluding health foods and health supplements, feeds, pet foodsincluding bait for fish, cosmetics, pharmaceuticals, and favorite foods.

The saccharides according to the present invention can be also used as aseasoning for sweetening various products, and In necessary, they can beused in combination with one or more other sweeteners such as a cornsyrup solid, glucose, fructose, lactosucrose, α,α-trehalose (aliastrehalose), α,β-trehalose (alias neotrehalose), β,β-trehalose, maltose,sucrose, isomerized sugar, honey, maple sugar. isomaltooligosaccharide,galactooligosaccharide, lactooligosaccharide, fructooligosaccharide,sorbitol, maltitol, lactitol, dihydrochalcone, stevioside, α-glycosylstevioside, rebaudioside, glycyrrhizin, L-aspartyl L-phenylalaninemethyl ester, sucralose, acesulfame K, saccharin, glycine, and alanine.If necessary, one or more fillers such as dextrins, starches, andlactose can be suitably used in combination.

The saccharides, particularly, those comprising the crystallineisomaltitol powder according to the present invention can be used alone,and optionally they can be used in combination with one or more ofappropriate fillers, excipients, binders, sweeteners to make them intodifferent shapes of granules, spheres, short rods, plates, cubes,tablets, films, or sheets.

The saccharides of the present invention have a sweetness that wellharmonize with other tastable substances having sour-, acid-, salty-,astringent-, delicious-, and bitter-tastes; and have a satisfactorilyhigh acid- and heat-tolerance. Thus, they can be favorably used tosweeten, improve the taste, or improve the quality of various foods andbeverages, for example, amino acids, peptides, soy sauce, powdered soysauce, miso, “funmatsu-miso” (a powdered miso), “moromi” (a refinedsake), “hishio” (a refined soy sauce), “furikake” (a seasoned fishmeal), mayonnaise, dressing, vinegar, “sanbai-zu” (a sauce of sugar, soysauce and vinegar), “funmatsu-sushi-su” (powdered vinegar for sushi),“chuka-no-moto” (an instant mix for Chinese dish), “tentsuyu” (a saucefor Japanese deep-fat fried food), “mentsuyu” (a sauce for Japanesevermicelli), sauce, catsup, “yakiniku-no-tare” (a sauce for Japanesegrilled meat), curry roux, instant stew mix, instant soup mix,“dashi-no-moto” (an instant stock mix), nucleotide seasonings, mixedseasoning, “mirin” (a sweet sake), “shin-mirin” (a synthetic mirin),table sugar, and coffee sugar. Also, the saccharide of the presentinvention can be arbitrarily used in “wagashi” (Japanese cakes) such as“senbei” (a rice cracker), “arare” (a rice cake cube), “okoshi” (amillet-and-rice cake), “mochi” (a rice paste) or the like, “manju” (abun with a bean-jam), “uiro” (a sweet rice jelly), “an” (a bean jam) orthe like, “yokan” (a sweet jelly of beans), “mizu-yokan” (a softadzuki-bean jelly), “kingyoku” (a kind of yokan), jelly, pao deCastella, and “amedama” (a Japanese toffee); Western confectioneriessuch as a bun, biscuit, cracker, cookie, pie, pudding, butter cream,custard cream, cream puff, waffle, sponge cake, doughnut, Yorkshirepudding, chocolate, chewing gum, caramel, and candy; frozen dessertssuch as an ice cream and sherbet; syrups such as a“kajitsu-no-syrup-zuke” (a preserved fruit) and “korimitsu” (a sugarsyrup for shaved ice); pastes such as a flour paste, peanut paste, fruitpaste, and spread; processed fruits and vegetables such as a jam,marmalade, “syrup-zuke” (fruit pickles), and “toka” (conserves); picklesand pickled products such as a “fukujin-zuke” (red colored radishpickles), “bettara-zuke” (a kind of whole fresh radish pickles),“senmai-zuke” (a kind of sliced fresh radish pickles), and “rakkyo-zuke”(pickled shallots); premixes for pickles and pickled products such as a“takuan-zuke-no-moto” (a premix for pickled radish), and“hakusai-zuke-no-moto” (a premix for fresh white rape pickles); meatproducts such as a ham and sausage; products of fish meat such as a fishham, fish sausage, “kamaboko” (a steamed fish paste), “chikuwa” (a kindof fish paste), and “tenpura” (a Japanese deep-fat fried fish paste);“chinmi” (relish) such as a “uni-no-shiokara” (a salted gut of seaurchin), “ika-no-shiokara” (a salted gut of squid), “su-konbu”(processed tangle), “saki-surume” (a dried squid strip), and“fugu-no-mirin-boshi” (a dried mirin-seasoned swellfish); “tsukudani”(foods boiled down in soy sauce) such as those of layer, edible wildplants, dried squid, small fish, and shellfish; daily dishes such as a“nimame” (cooked beans), potato salad, and “konbu-maki” (a tangle roll);milk products such as a yogurt and cheese; canned and bottled productssuch as those of meat, fish meat, fruit, and vegetables; alcoholicbeverages such as sake, distilled spirit, shochu-based beverage,synthetic sake, liqueur, cocktail, and others; soft drinks such as acoffee, tea, cocoa, juice, isotonic beverage, carbonated beverage, sourmilk beverage, and beverage containing lactic acid bacteria; instantfood products such as an instant pudding mix, instant hot cake mix,“sokuseki-shiruko” (an instant mix of adzuki-bean soup with rice cake),and instant soup mix; and other foods and beverages such as solid foodsfor babies, foods for therapy, health/tonic drinks, peptide foods,frozen foods, and health foods. The saccharide of the present inventioncan be arbitrarily used to improve the taste preference of feeds andfoods for animals and pets such as domestic animals, poultry, honeybees, silk warms, fishes, crustaceans including shrimps/prawns/lobsters,and crabs. In addition, the saccharides of the present invention can beused as a sweetener for solid products such as a tobacco, cigarette,tooth paste, lipstick/rouge, lip cream, internal liquid medicine,tablet, troche, cod liver oil in the form of a drop, cachou, oralrefrigerant, or gargle. Also the saccharides can be used in the aboveproducts as a taste-improving agent, flavoring substance,quality-improving agent, stabilizer, or moisture-retaining agent.

The saccharides of the present invention are sugar alcohols which do notcause the Maillard reaction because of their non-reducibility.Therefore, the saccharides have no fear of deteriorating effectiveingredients such as amino compounds and can be incorporated as aquality-improving agent and/or stabilizer into health foods andpharmaceuticals, which have effective ingredients, active components, orphysiologically active substances, to obtain stabilized, high qualityhealth foods or pharmaceuticals in the form of a liquid, paste, orsolid. Examples of the above-identified effective ingredients andbiologically active substances are lymphokines such as α-, β- andγ-interferons, tumor necrosis factor-α (TNF-α), tumor necrosis factor-β(TNF-β), macrophage migration inhibitory factor, colony-stimulatingfactor, transfer factor, and interleukins; hormones such as insulin,growth hormone, prolactin, erythropoietin, and follicle-stimulatinghormone; biological preparations such as BCG vaccine, Japaneseencephalitis vaccine, measles vaccine, live polio vaccine, smallpoxvaccine, tetanus toxoid, Trimeresurus antitoxin, and humanimmunoglobulin; antibiotics such as penicillin, erythromycin,chloramphenicol, tetracycline, streptomycin, and kanamycin sulfate;vitamins such as thiamine, riboflavin, L-ascorbic acid, α-glycosylascorbic acid, cod liver oil, carotenoid, ergosterol, tocopherol, rutin,α-glycosyl rutin, naringin, α-glycosyl naringin, hesperidin, andα-glycosyl hesperidin; enzymes such as lipase, elastase, urokinase,protease, β-amylase, isoamylase, glucanase, and lactase; extracts suchas ginseng extract, bamboo leaf extract, Japanese plum extract, pineleaf extract, snapping turtle extract, chlorella extract, aloe extract,and propolis extract; viable microorganisms such as viruses, lactic acidbacteria, and yeasts; and royal jelly.

The methods for incorporating the saccharides of the present inventioninto the aforesaid compositions are those which can incorporate thesaccharides into the compositions before completion of theirprocessings, and which can be appropriately selected among the followingconventional methods; mixing, dissolving, melting, soaking, penetrating,dispersing, applying, coating, spraying, injecting, crystallizing, andsolidifying. The amount of the saccharides to be incorporated into eachof the above compositions is usually in an amount of at least 0.1%,desirably, at least 1%, and more desirably, 2 to 99.9% by weight of eachof the compositions.

The following experiments explain the process for producing isomaltoseand isomaltitol according to the present invention:

EXPERIMENT 1 Preparation of Non-Reducing Cyclotetrasaccharide byCulturing

A liquid medium, consisting of 5% (w/v) of “PINE-DEX #1”, a partialstarch hydrolyzate commercialized by Matsutani Chemical Ind., Tokyo,Japan, 1.5% (w/v) of “ASAHIMEAST™”, a yeast extract commercialized byAsahi Breweries, Ltd., Tokyo, Japan, 0.1% (w/v) of dipotassiumphosphate, 0.06% (w/v) of sodium phosphate dodecahydrate, 0.05% (w/v)magnesium sulfate heptahydrate, and water, was placed in a 500-mlErlenmeyer flask in an amount of 100 ml, sterilized by autoclaving at121° C. for 20 min, cooled, and then seeded with a stock culture ofBacillus globisporus C9 strain (FERM BP-7143), followed by culturingunder rotary-shaking conditions at 27° C. and 230 rpm for 48 hours andcentrifuging the resulting culture to remove cells and to obtain asupernatant. The supernatant was autoclaved at 120° C. for 15 min andthen cooled, and the resulting insoluble substances were removed bycentrifugation to obtain a supernatant.

To examine the saccharides in the supernatant, they were separated bydeveloping twice on silica gel thin-layer chromatography (abbreviated as“TLC” hereinafter) using, as a developer, a mixture solution ofn-butanol, pyridine, and water (=6:4:1 by volume), and, as a thin-layerplate, “KIESELGEL™ 60”, an aluminum plate (20×20 cm) for TLCcommercialized by Merck & Co., Inc., Rahway, USA. The coloration of theseparated total sugars by the sulfuric acid-methanol method and that ofthe reducing saccharides by the diphenylamine-aniline method detected anon-reducing saccharide positive at an Rf value of about 0.31 on theformer detection method but negative on the latter detection method.

About 90 ml of the supernatant obtained in the above was adjusted to pH5.0 and heated to 45° C. and then incubated for 24 hours after admixedwith 1,500 units per gram of solids of “TRANSGLUCOSIDASE L AMANO™”, anα-glucosidase specimen commercialized by Amano Pharmaceutical Co., Ltd.,Aichi, Japan; and 75 units per gram of solids of a glucoamylasecommercialized by Nagase Biochemicals, Ltd., Kyoto, Japan. Thereafter,the resulting culture was adjusted to pH 12 by the addition of sodiumhydroxide and boiled for two hours to decompose the remaining reducingsugars. After removing insoluble substances by filtration, the resultingsolution was decolored and desalted with “DIAION PK218™” and “DIAIONWA30™”, cation exchange resins commercialized by Mitsubishi ChemicalIndustries, Ltd., Tokyo, Japan; and further desalted with “DIAIONSK-1B™”, commercialized by Mitsubishi Chemical Industries, Ltd., Tokyo,Japan, and “AMBERLITE IRA411™”, an anion exchange resin commercializedby Japan Organo Co., Ltd., Tokyo, Japan. The resulting solution wasdecolored with an activated charcoal, membrane filtered, concentrated byan evaporator, and lyophilized in vacuo to obtain about 0.6 g, d.s.b.,of a saccharide powder. The analysis of the saccharide powder onhigh-performance liquid chromatography (abbreviated as “HPLC”hereinafter) detected a single peak at an elution time of 10.84 min asshown in FIG. 1, and revealed that it had a purity of as high as 99.9%or higher. The above HPLC was run using “SHOWDEX KS-801™ column”, ShowaDenko K.K., Tokyo, Japan, at a column temperature of 60° C. and a flowrate of 0.5 ml/min of water, and “RI-8012”, a differential refractometercommercialized by Tosoh Corporation, Tokyo, Japan. When measured forreducing power of the saccharide on the Somogyi-Nelson's method, thereducing power was below a detectable level, meaning that the saccharidewas substantially a non-reducing saccharide.

EXPERIMENT 2 Structure Analysis of Non-reducing Saccharide

Fast atom bombardment mass spectrometry (called “FAB-MS”) of anon-reducing saccharide, obtained by the method in Experiment 1,significantly detected a proton-addition-molecular ion with a massnumber of 649, meaning that the saccharide had a mass number of 648.According to conventional manner, the saccharide was hydrolyzed withsulfuric acid and then analyzed for sugar composition on gaschromatography. As a result, D-glucose was detected only, revealing thatthe saccharide was composed of D-glucose molecules or acyclotetrasaccharide composed of four D-glucose molecules based on theabove mass number. Nuclear magnetic resonance analysis (called “NMR”) ofthe saccharide gave a ¹H-NMR spectrum as shown in FIG. 2 and a ¹³C-NMRspectrum as shown in FIG. 3, and these spectra were compared with thoseof conventional saccharides, revealing that the spectra were coincidedwith those of a non-reducing cyclic saccharide,cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→}disclosed in “European Journal of Biochemistry”, pp. 641-648 (1994). Thedata confirmed that the saccharide obtained in this experiment is acyclotetrasaccharide as shown in FIG. 4, i.e.,cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→}.

EXPERIMENT 3 Production of α-isomaltosylglucosaccharide-forming Enzymefrom Bacillus globisporus C9 Strain

A liquid culture medium, consisting of 4.0% (w/v) of “PINE-DEX #4™”, apartial starch hydrolyzate commercialized by Matsutani Chemical Ind.,Tokyo, Japan, 1.8% (w/v) of “ASAHIMEAST™”, a yeast extractcommercialized by Asahi Breweries, Ltd., Tokyo, Japan, 0.1% (w/v) ofdipotassium phosphate, 0.06% (w/v) of sodium phosphate dodecahydrate,0.05% (w/v) magnesium sulfate heptahydrate, and water, was placed in500-ml Erlenmeyer flasks in an amount of 100 ml each, sterilized byautoclaving at 121° C. for 20 min, cooled, and then seeded with a stockculture of Bacillus globisporus C9 strain (FERM BP-7143), followed byculturing under rotary-shaking conditions at 27° C. and 230 rpm for 48hours for a seed culture. About 20 L of a fresh preparation of the sameliquid culture medium as used in the above seed culture were placed in a30-L fermentor, sterilized by heating, and then cooled to 27° C. andinoculated with 1% (v/v) of the seed culture, followed by culturing at27° C. and pH 6.0 to 8.0 for 48 hours under aeration-agitationconditions. After completion of the culture, the resulting culture,which had about 0.45 unit/ml of α-isomaltosylglucosaccharide-formingenzyme, about 1.5 units/ml of α-isomaltosyl-transferring enzyme, andabout 0.95 unit/ml of a cyclotetrasaccharide-forming activity, wascentrifuged at 10,000 rpm for 30 min to obtain about 18 L of asupernatant. When measured for enzymatic activity, the supernatantcontained about 0.45 unit/ml of α-isomaltosylglucosaccharide-formingenzyme, i.e., a total enzymatic activity of about 8,110 units; about 1.5units/ml of α-isomaltosyl-transferring enzyme, i.e., a total enzymaticactivity of about 26,900 units; and about 0.95 unit/ml ofcyclotetrasaccharide-forming enzyme, i.e., a total enzymatic activity ofabout 17,100 units. These activities were assayed as follows: Theactivity of α-isomaltosylglucosaccharide-forming enzyme was assayed bydissolving maltotriose in 100 mM acetate buffer (pH 6.0) to give aconcentration of 2% (w/v) for a substrate solution, adding a 0.5 ml ofan enzyme solution to a 0.5 ml of the substrate solution, enzymaticallyreacting the mixture solution at 35° C. for 60 min, suspending theenzymatic reaction by boiling the solution for 10 min, and quantifyingmaltose, among the isomaltosyl maltose and maltose formed mainly in thereaction mixture, on HPLC disclosed in Experiment 1. One unit activityof α-isomaltosylglucosaccharide-forming enzyme is defined as the enzymeamount that forms one micromole of maltose per minute under the aboveenzymatic reaction conditions. Throughout the specification, theenzymatic activity of α-isomaltosylglucosaccharide-forming enzyme meansthe unit(s) assayed as above.

The activity of α-isomaltosyl-transferring enzyme was assayed bydissolving panose in 100 mM acetate buffer (pH 6.0) to give aconcentration of 2% (w/v) for a substrate solution, adding a 0.5 ml ofan enzyme solution to 0.5 ml of the substrate solution, enzymaticallyreacting the mixture solution at 35° C. for 30 min, suspending theenzymatic reaction by boiling the solution for 10 min, and quantifyingglucose, among the cyclotetrasaccharide and glucose formed mainly in thereaction mixture, by the glucose oxidase method. One unit activity ofα-isomaltosyl-transferring enzyme is defined as the enzyme amount thatforms one micromole of glucose per minute under the above enzymaticreaction conditions. Throughout the specification, the enzymaticactivity of α-isomaltosyl-transferring enzyme means the unit(s) assayedas above.

The cyclotetrasaccharide-forming activity was assayed by dissolving“PINE-DEX #100™”, a partial starch hydrolyzate commercialized byMatsutani Chemical Ind., Tokyo, Japan, in 50 mM acetate buffer (pH 6.0)to give a concentration of 2% (w/v) for a substrate solution, adding 0.5ml of an enzyme solution to 0.5 ml of the substrate solution,enzymatically reacting the mixture solution at 35° C. for 60 min,suspending the enzymatic reaction by heating the solution at 100° C. for10 min, and then further adding to the resulting solution one milliliterof 50 mM acetate buffer (pH 5.0) with 70 units/ml of “TRANSGLUCOSIDASE LAMANO™”, an α-glucosidase commercialized by Amano Pharmaceutical Co.,Ltd., Aichi, Japan, and 27 units/ml of glucoamylase, commercialized byNagase Biochemicals, Ltd., Kyoto, Japan, followed by incubating themixture at 50° C. for 60 min, inactivating the remaining enzymes byheating at 100° C. for 10 min, and quantifying cyclotetrasaccharide onHPLC described in Experiment 1. One unit activity ofcyclotetrasaccharide-forming enzyme is defined as the enzyme amount thatforms one micromole of cyclotetrasaccharide per minute under the aboveenzymatic reaction conditions. Throughout the specification, theactivity of cyclotetrasaccharide-forming enzyme means the unit(s)assayed as above.

EXPERIMENT 4 Preparation of Enzyme from Bacillus globisporus C9 StrainEXPERIMENT 4-1

About 18 L of the supernatant in Experiment 3 were salted out in 80%saturated ammonium sulfate and allowed to stand at 4° C. for 24 hours,and the formed sediments were collected by centrifugation at 10,000 rpmfor 30 min, dissolved in 10 mM phosphate buffer (pH 7.5), and dialyzedagainst a fresh preparation of the same buffer to obtain about 400 ml ofa crude enzyme solution with 8,110 units of anα-isomaltosylglucosaccharide-forming activity, 24,700 units of anα-isomaltosyl-transferring activity, and about 15,600 units of acyclotetrasaccharide-forming activity. The crude enzyme solution wassubjected to ion-exchange chromatography using 1,000 ml of “SEPABEADSFP-DA13 ™” gel, an ion-exchange resin commercialized by MitsubishiChemical Industries, Ltd., Tokyo, Japan. Theα-isomaltosylglucosaccharide-forming enzyme and cyclotetrasaccharidewere eluted as non-adsorbed fractions without adsorbing on theion-exchange resin. The resulting enzyme solution was dialyzed against10 mM phosphate buffer (pH 7.0) with 1 M ammonium sulfate, and thedialyzed solution was centrifuged to remove insoluble impurities, andsubjected to affinity chromatography using 500 ml of “SEPHACRYL HRS-200™”, a gel commercialized by Amersham Corp., Div., AmershamInternational, Arlington Heights, Ill., USA. Enzymatically activecomponents adsorbed on the gel and, when sequentially eluted with alinear gradient decreasing from 1 M to 0 M of ammonium sulfate and alinear gradient increasing from 0 mM to 100 mM of maltotetraose, theα-isomaltosyl-transferring enzyme and theα-isomaltosylglucosaccharide-forming enzyme were separately eluted,i.e., the former was eluted with the linear gradient of ammonium sulfateat about 0 M and the latter was eluted with the linear gradient ofmaltotetraose at about 30 mM. Thus, fractions with anα-isomaltosyl-transferring activity and those with anα-isomaltosylglucosaccharide-forming activity were separatory collected.No cyclotetrasaccharide-forming activity was found in any of the abovefractions but found in their mixture solution, and the fact revealedthat the activity of forming cyclotetrasaccharide from partial starchhydrolyzates was exerted by the coaction of the activities of the abovetwo types of enzymes.

Methods for separately purifying α-isomaltosylglucosaccharide-formingenzyme and α-isomaltosyl-transferring enzyme are described below:

EXPERIMENT 4-2 Purification of α-isomaltosylglucosaccharide-formingEnzyme

Factions of α-isomaltosylglucosaccharide-forming enzyme, obtained inExperiment 4-1, were pooled and dialyzed against 10 mM phosphate buffer(pH 7.0) containing 1 M ammonium sulfate. The dialyzed solution wascentrifuged to remove insoluble impurities, and the resultingsupernatant was fed to hydrophobic chromatography using 350 ml of“BUTYL-TOYOPEARL 650 M™”, a gel for hydrophobic chromatographycommercialized by Tosoh Corporation, Tokyo, Japan. The enzyme wasadsorbed on the gel and eluted at about 0.3 M ammonium sulfate wheneluted with a linear gradient decreasing from 1 M to 0 M of ammoniumsulfate, followed by collecting fractions with the enzyme activity. Thefractions were pooled and dialyzed against 10 mM phosphate buffer (pH7.0) containing 1 M ammonium sulfate. The resulting dialyzed solutionwas centrifuged to remove insoluble impurities and fed to affinitychromatography using “SEPHACRYL HR S-200™” gel to purify the enzyme. Theamount of enzyme activity, specific activity, and yield ofα-isomaltosylglucosaccharide-forming enzyme in each purification stepare in Table 1.

TABLE 1 Specific activity Enzyme* activity of enzyme* Yield Purificationstep (unit) (unit/mg protein) (%) Culture supernatant 8,110 0.12 100Dialyzed solution after 7,450 0.56 91.9 salting out with ammoniumsulfate Eluate from ion-exchange 5,850 1.03 72.1 column chromatographyEluate from affinity 4,040 8.72 49.8 column chromatography Eluate fromhydrophobic 3,070 10.6 37.8 column chromatography Eluate from affinity1,870 13.6 23.1 column chromatography Note: The symbol “*” meansα-isomaltosylglucosaccharide-forming enzyme.

The finally purified α-isomaltosylglucosaccharide-forming enzymespecimen was examined for purity on gel electrophoresis using a 7.5%(w/v) polyacrylamide gel and detected on the gel as a single proteinband, i.e., a high purity enzyme specimen.

EXPERIMENT 4-3 Purification of α-isomaltosyl-transferring Enzyme

Fractions with α-isomaltosyl-transferring enzyme, which had beenseparated from the fractions with α-isomaltosylglucosaccharide-formingenzyme by affinity chromatography in Experiment 4-1, were pooled anddialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammoniumsulfate. The resulting dialyzed solution was centrifuged to removeinsoluble impurities and subjected to affinity chromatography using 350ml of “BUTYL-TOYOPEARL 650M”, a gel for hydrophobic chromatographycommercialized by Tosoh Corporation, Tokyo, Japan, to purify the enzyme.The enzyme was adsorbed on the gel and eluted therefrom at aconcentration of about 0.3 M ammonium sulfate when eluted with a lineargradient decreasing from 1 M to 0 M of ammonium sulfate, followed bycollecting fractions with the enzyme activity. The fractions were pooledand dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 Mammonium sulfate. The resulting dialyzed solution was centrifuged toremove insoluble impurities and fed to affinity chromatography using“SEPHACRYL HR S-200” gel to purify the enzyme. The amount of enzymeactivity, specific activity, and yield of α-isomaltosyl-transferringenzyme in each purification step are in Table 2.

TABLE 2 Specific activity Enzyme* activity of enzyme* Yield Purificationstep (unit) (unit/mg protein) (%) Culture supernatant 26,900 0.41 100Dialyzed solution after 24,700 1.85 91.8 salting out with ammoniumsulfate Eluate from ion-exchange 19,400 3.41 72.1 column chromatographyEluate from affinity 13,400 18.6 49.8 column chromatography Eluate fromhydrophobic 10,000 21.3 37.2 column chromatography Eluate from affinity6,460 26.9 24.0 column chromatography Note: The symbol “*” means theα-isomaltosyl-transferring enzyme.

EXPERIMENT 5 Property of α-isomaltosylglucosaccharide-forming Enzyme andα-isomaltosyl-transferring Enzyme EXPERIMENT 5-1 Property ofα-isomaltosylglucosaccharide-forming Enzyme

A purified specimen of α-isomaltosylglucosaccharide-forming enzyme,obtained by the method in Experiment 4-2, was subjected to SDS-PAGEusing a 7.5% (w/v) of polyacrylamide gel and then determined formolecular weight by comparing with the dynamics of standard molecularmarkers electrophoresed in parallel, commercialized by Japan Bio-RadLaboratories Inc., Tokyo, Japan, revealing that the enzyme had amolecular weight of about 140,000±20,000 daltons.

A fresh preparation of the above purified specimen was subjected toisoelectrophoresis using a gel containing 2% (w/v) ampholinecommercialized by Amersham Corp., Div., Amersham International,Arlington Heights, Ill., USA, and then measured for pHs of protein bandsand gel to determine the isoelectric point of the enzyme, revealing thatthe enzyme had an isoelectric point of about 5.2±0.5. The influence oftemperature and pH on the activity ofα-isomaltosylglucosaccharide-forming enzyme was examined in accordancewith the assay for its enzyme activity, where the influence oftemperature was examined in the presence or the absence of 1 mM Ca²⁺.These results are in FIG. 5 (influence of temperature) and FIG. 6(influence of pH). The optimum temperature of the enzyme was about 40°C. (in the absence of Ca²⁺) and about 45° C. (in the presence of 1 mMCa²⁺) when incubated at pH 6.0 for 60 min, and the optimum pH of theenzyme was about 6.0 to about 6.5 when incubated at 35° C. for 60 min.The thermal stability of the enzyme was determined by incubating thetesting enzyme solutions in the form of 20 mM acetate buffer (pH 6.0) atprescribed temperatures for 60 min in the presence or the absence of 1mM Ca²⁺, cooling the resulting enzyme solutions with water, and assayingthe remaining enzyme activity of each solution. The pH stability of theenzymes was determined by keeping the testing enzyme solutions in theform of an appropriate 50 mM buffer having a prescribed pH at 4° C. for24 hours, adjusting the pH of each solution to 6.0, and assaying theremaining enzyme activity of each solution. These results arerespectively in FIG. 7 (thermal stability) and FIG. 8 (pH stability). Asa result, the enzyme had thermal stability of up to about 35° C. in theabsence of Ca²⁺ and about 40° C. in the presence of 1 mM Ca²⁺, and pHstability of about 4.5 to about 9.0.

The influence of metal ions on the activity ofα-isomaltosylglucosaccharide-forming enzyme was examined in the presenceof 1 mM of each metal-ion according to the assay for its enzymeactivity. The results are in Table 3.

TABLE 3 Metal Relative activity Metal Relative activity ion (%) ion (%)None 100 Hg²⁺ 4 Zn²⁺ 92 Ba²⁺ 65 Mg²⁺ 100 Sr²⁺ 80 Ca²⁺ 115 Pb²⁺ 103 Co²⁺100 Fe²⁺ 98 Cu²⁺ 15 Fe³⁺ 97 Ni²⁺ 98 Mn²⁺ 111 Al³⁺ 99 EDTA 20

As evident form the results in Table 3, the enzyme activity was stronglyinhibited by Hg²⁺, Cu²⁺, and EDTA, and it was also inhibited by Ba²⁺ andSr²⁺. It was also found that the enzyme was activated by Ca²⁺ and Mn²⁺.

Amino acid analysis on the N-terminal amino acid sequence of the enzymeby “PROTEIN SEQUENCER MODEL 473A”, an apparatus of Applied Biosystems,Inc., Foster City, USA, revealed that the enzyme had a partial aminoacid sequence of SEQ ID NO:1, i.e.,tyrosine-valine-serine-serine-leucine-glycine-asparagine-leucine-isoleucinein the N-terminal region.

EXPERIMENT 5-2 Property of α-isomaltosyl-transferring Enzyme

A purified specimen of α-isomaltosyl-transferring enzyme, obtained bythe method in Experiment 4-3, was subjected to SDS-PAGE using a 7.5%(w/v) of polyacrylamide gel and then determined for molecular weight bycomparing with the dynamics of standard molecular markerselectrophoresed in parallel, commercialized by Japan Bio-RadLaboratories Inc., Tokyo, Japan, revealing that the enzyme had amolecular weight of about 112,000±20,000 daltons.

A fresh preparation of the above purified specimen was subjected toisoelectrophoresis using a gel containing 2% (w/v) ampholinecommercialized by Amersham Corp., Div., Amersham International,Arlington Heights, Ill., USA, and then measured for pHs of protein bandsand gel to determine the isoelectric point of the enzyme, revealing thatthe enzyme had an isoelectric point of about 5.5±0.5.

The influence of temperature and pH on the activity ofα-isomaltosyl-transferring enzyme was examined in accordance with theassay for its enzyme activity. These results are in FIG. 9 (influence oftemperature) and FIG. 10 (influence of pH). The optimum temperature ofthe enzyme was about 45° C. when incubated at pH 6.0 for 30 min, and theoptimum pH of the enzyme was about 6.0 when incubated at 35° C. for 30min. The thermal stability of the enzyme was determined by incubatingthe testing enzyme solutions in the form of 20 mM acetate buffer (pH6.0) at prescribed temperatures for 60 min, cooling the resulting enzymesolutions with water, and assaying the remaining enzyme activity of eachsolution. The pH stability of the enzyme was determined by keeping thetesting enzyme solutions in the form of an appropriate 50 mM bufferhaving a prescribed pH at 4° C. for 24 hours, adjusting the pH of eachsolution to 6.0, and assaying the remaining enzyme activity of eachsolution. These results are respectively in FIG. 11 (thermal stability)and FIG. 12 (pH stability). As a result, the enzyme had thermalstability of up to about 40° C. and pH stability of about 4.0 to about9.0.

The influence of metal ions on the activity ofα-isomaltosyl-transferring enzyme was examined in the presence of 1 mMof each metal-ion according to the assay for its enzyme activity. Theresults are in Table 4.

TABLE 4 Relative activity Metal Relative activity Metal ion (%) ion (%)None 100 Hg²⁺ 1 Zn²⁺ 88 Ba²⁺ 102 Mg²⁺ 98 Sr²⁺ 101 Ca²⁺ 101 Pb²⁺ 89 Co²⁺103 Fe²⁺ 96 Cu²⁺ 57 Fe³⁺ 105 Ni²⁺ 102 Mn²⁺ 106 Al³⁺ 103 EDTA 104

As evident form the results in Table 4, the enzyme activity was stronglyinhibited by Hg²⁺, and it was also inhibited by Cu²⁺. It was also foundthat the enzyme was not activated by Ca²⁺ and not inhibited by EDTA.

Amino acid analysis on the N-terminal amino acid sequence of the enzymeby “PROTEIN SEQUENCER MODEL 473A”, an apparatus of Applied Biosystems,Inc., Foster City, USA, revealed that the enzyme had a partial aminoacid sequence of SEQ. ID NO:2, i.e, isoleucine-asparticacid-glycine-valine-tyrosine-histidine-alanine-proline-asparagine-glycinein the N-terminal region.

EXPERIMENT 6 Production of α-isomaltosylglucosaccharide-forming Enzymefrom Bacillus globisporus C11 Strain

A liquid nutrient culture medium, consisting of 4.0% (w/v) of “PINE-DEX#4”, a partial starch hydrolyzate, 1.8% (w/v) of “ASAHIMEAST”, a yeastextract, 0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodiumphosphate dodecahydrate, 0.05% (w/v) magnesium sulfate heptahydrate, andwater, was placed in 500-ml Erlenmeyer flasks in a volume of 100 mleach, autoclaved at 121° C. for 20 minutes to effect sterilization,cooled, inoculated with a stock culture of Bacillus globisporus C11strain (FERM BP-7144), and incubated at 27° C. for 48 hours under rotaryshaking conditions of 230 rpm. The resulting cultures were pooled andused as a seed culture. About 20 L of a fresh preparation of the samenutrient culture medium as used in the above culture were placed in a30-L fermentor, sterilized by heating, cooled to 27° C., inoculated with1% (v/v) of the seed culture, and incubated for about 48 hours whilestirring under aeration-agitation conditions at 27° C. and a pH of 6.0to 8.0. The resultant culture, having about 0.55 unit/ml of anα-isomaltosylglucosaccharide-forming activity, about 1.8 units/ml of anα-isomaltosyl-transferring activity, and about 1.1 units/ml of acyclotetrasaccharide-forming activity, was centrifuged at 10,000 rpm for30 min to obtain about 18 L of a supernatant. Measurement of thesupernatant revealed that it had about 0.51 unit/ml of anα-isomaltosylglucosaccharide-forming enzyme activity, i.e., a totalenzyme activity of about 9,180 units; about 1.7 units/ml of anα-isomaltosyl-transferring enzyme activity, i.e., a total enzymeactivity of about 30,400 units; and about 1.1 units/ml of acyclotetrasaccharide-forming enzyme activity, i.e., a total enzymeactivity of about 19,400 units.

EXPERIMENT 7 Preparation of enzyme from Bacillus globisporus C11 StrainEXPERIMENT 7-1 Purification of Enzyme from Bacillus globisporus C11Strain

Eighteen litters of the supernatant, obtained in Experiment 6, weresalted out in an 80% saturated ammonium sulfate solution and allowed tostand at 4° C. for 24 hours. Then, the salted out sediments werecollected by centrifugation at 10,000 for 30 min, dissolved in 10 mMphosphate buffer (pH 7.5), dialyzed against a fresh preparation of thesame buffer as used in the above to obtain about 416 ml of a crudeenzyme solution. The crude enzyme solution was revealed to have 8,440units of an α-isomaltosylglucosaccharide-forming enzyme activity, about28,000 units of an α-isomaltosyl-transferring enzyme activity, and about17,700 units of a cyclotetrasaccharide-forming enzyme activity. Whensubjected to ion-exchange chromatography using “SEPABEADS FP-DA13” gel,disclosed in Experiment 4-1, the above three types of enzymes wereeluted as non-adsorbed fractions without adsorbing on the gel. Thenon-adsorbed fractions with those enzymes were pooled and dialyzedagainst 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate,and the dialyzed solution was centrifuged to remove insolubleimpurities. The resulting supernatant was fed to affinity chromatographyusing 500 ml of “SEPHACRYL HR S-200” gel to purify the enzyme. Activeenzymes were adsorbed on the gel and sequentially eluted with a lineargradient decreasing from 1 M to 0 M of ammonium sulfate and a lineargradient increasing from 0 mM to 100 mM of maltotetraose, followed bycollecting separate elutions of α-isomaltosyl-transferring enzyme andα-isomaltosylglucosaccharide-forming enzyme, respectively, where theformer enzyme was eluted with the linear gradient of ammonium sulfate ata concentration of about 0.3 M and the latter enzyme was eluted with alinear gradient of maltotetraose at a concentration of about 30 mM.Therefore, fractions with the α-isomaltosylglucosaccharide-formingenzyme and those with the α-isomaltosyl-transferring enzyme wereseparately collected. Similarly as in the case of Bacillus globisporusC9 strain in Experiment 4, it was found that nocyclotetrasaccharide-forming activity was found in any fraction in thiscolumn chromatography, and that an enzyme mixture solution of bothfractions of α-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme showed a cyclotetrasaccharide-formingenzyme activity, revealing that the activity of formingcyclotetrasaccharide from partial starch hydrolyzates was exerted incollaboration with the enzyme activities of the two types of enzymes.

Methods for separately purifying α-isomaltosylglucosaccharide-formingenzyme and α-isomaltosyl-transferring enzyme are explained below:

EXPERIMENT 7-2 Purification of α-isomaltosylglucosaccharide-formingEnzyme

A faction of α-isomaltosylglucosaccharide-forming enzyme, obtained inExperiment 7-1, was dialyzed against 10 mM phosphate buffer (pH 7.0)containing 1 M ammonium sulfate. The dialyzed solution was centrifugedto remove insoluble impurities, and the resulting supernatant was fed tohydrophobic chromatography using 350 ml of “BUTYL-TOYOPEARL 650 M”, agel commercialized by Tosoh Corporation, Tokyo, Japan. The enzymeadsorbed on the gel was eluted at about 0.3 M ammonium sulfate wheneluted with a linear gradient decreasing from 1 M to 0 M of ammoniumsulfate, followed by collecting fractions with the enzyme activity. Thefractions were pooled and dialyzed against 10 mM phosphate buffer (pH7.0) containing 1 M ammonium sulfate. The resulting dialyzed solutionwas centrifuged to remove insoluble impurities and fed to affinitychromatography using “SEPHACRYL HR S-200” gel to purify the enzyme. Theamount of enzyme activity, specific activity, and yield of theα-isomaltosylglucosaccharide-forming enzyme in each purification stepare in Table 5.

TABLE 5 Specific activity Enzyme* activity of enzyme* Yield Purificationstep (unit) (unit/mg protein) (%) Culture supernatant 9,180 0.14 100Dialyzed solution after 8,440 0.60 91.9 salting out with ammoniumsulfate Eluate from ion-exchange 6,620 1.08 72.1 column chromatographyEluate from affinity 4,130 8.83 45.0 column chromatography Eluate fromhydrophobic 3,310 11.0 36.1 column chromatography Eluate from affinity2,000 13.4 21.8 column chromatography Note: The symbol “*” meansα-isomaltosylglucosaccharide-forming enzyme.

The finally purified α-isomaltosylglucosaccharide-forming enzymespecimen was assayed for purity on gel electrophoresis using a 7.5%(w/v) polyacrylamide gel and detected on the gel as a single proteinband, meaning a high purity enzyme specimen.

EXPERIMENT 7-3 Purification of α-isomaltosyl-transferring Enzyme

A faction of α-isomaltosyl-transferring enzyme, which had been separatedfrom a fraction of α-isomaltosylglucosaccharide-forming enzyme by theaffinity chromatography in Experiment 7-1, was dialyzed against 10 mMphosphate buffer (pH 7.0) containing 1 M ammonium sulfate. The dialyzedsolution was centrifuged to remove insoluble impurities, and theresulting supernatant was fed to hydrophobic chromatography using 350 mlof “BUTYL-TOYOPEARL 650 M”, a gel commercialized by Tosoh Corporation,Tokyo, Japan. The enzyme adsorbed on the gel and then it was eluted atabout 0.3 M ammonium sulfate when eluted with a linear gradientdecreasing from 1 M to 0 M of ammonium sulfate, followed by collectingfractions with the enzyme activity. The fractions were pooled anddialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammoniumsulfate. The resulting dialyzed solution was centrifuged to removeinsoluble impurities and fed to affinity chromatography using “SEPHACRYLHR S-200” gel to purify the enzyme. The amount of enzyme activity,specific activity, and yield of the α-isomaltosyl-transferring enzyme ineach purification step are in Table 6.

TABLE 6 Specific activity Enzyme* activity of enzyme* Yield Purificationstep (unit) (unit/mg protein) (%) Culture supernatant 30,400 0.45 100Dialyzed solution after 28,000 1.98 92.1 salting out with ammoniumsulfate Eluate from ion-exchange 21,800 3.56 71.7 column chromatographyEluate from affinity 13,700 21.9 45.1 column chromatography Eluate fromhydrophobic 10,300 23.4 33.9 column chromatography Eluate from affinity5,510 29.6 18.1 column chromatography Note: The symbol “*” meansα-isomaltosyl-transferring enzyme.

EXPERIMENT 8 Property of α-isomaltosylglucosaccharide-forming Enzyme andα-isomaltosyl-transferring Enzyme EXPERIMENT 8-1 Property ofα-isomaltosylglucosaccharide-forming Enzyme

A purified specimen of α-isomaltosylglucosaccharide-forming enzyme,obtained by the method in Experiment 7-2, was subjected to SDS-PAGEusing a 7.5% (w/v) of polyacrylamide gel and then determined formolecular weight by comparing with the dynamics of standard molecularmarkers electrophoresed in parallel, commercialized by Japan Bio-RadLaboratories Inc., Tokyo, Japan, revealing that the enzyme had amolecular weight of about 137,000±20,000 daltons.

A fresh preparation of the same purified specimen as used in the abovewas subjected to isoelectrophoresis using a gel containing 2% (w/v)ampholine commercialized by Amersham Corp., Div., AmershamInternational, Arlington Heights, Ill., USA, and then measured for pHsof protein bands and gel to determine the isoelectric point of theenzyme, revealing that the enzyme had an isoelectric point of about5.2±0.5.

The influence of temperature and pH on the activity ofα-isomaltosylglucosaccharide-forming enzyme was examined in accordancewith the assay for its enzyme activity, where the influence oftemperature was examined in the presence or the absence of 1 mM Ca²⁺.These results are in FIG. 13 (influence of temperature) and FIG. 14(influence of pH). The optimum temperature of the enzyme was about 45°C. in the absence of Ca²⁺ and about 50° C. in the presence of 1 mM Ca²⁺when incubated at pH 6.0 for 60 min. The optimum pH of the enzyme wasabout 6.0 when incubated at 35° C. for 60 min. The thermal stability ofthe enzyme was determined by incubating the testing enzyme solutions inthe form of 20 mM acetate buffer (pH 6.0) in the presence or the absenceof 1 mM Ca²⁺ at prescribed temperatures for 60 min, cooling theresulting enzyme solutions with water, and assaying the remaining enzymeactivity of each solution. The pH stability of the enzyme was determinedby keeping the testing enzyme solutions in the from of 50 mM buffershaving prescribed pHs at 4° C. for 24 hours, adjusting the pH of eachsolution to 6.0, and assaying the remaining enzyme activity of eachsolution. These results are respectively in FIG. 15 (thermal stability)and FIG. 16 (pH stability). As a result, the enzyme had thermalstability of up to about 40° C. in the absence of Ca²⁺ and up to about45° C. in the presence of 1 mM Ca²⁺. The pH stability of enzyme wasabout 5.0 to about 10.0.

The influence of metal ions on the activity ofα-isomaltosyl-transferring enzyme was examined in the presence of 1 mMof each metal-ion according to the assay for its enzyme activity. Theresults are in Table 7.

TABLE 7 Relative activity Metal Relative activity Metal ion (%) ion (%)None 100 Hg²⁺ 4 Zn²⁺ 91 Ba²⁺ 65 Mg²⁺ 98 Sr²⁺ 83 Ca²⁺ 109 Pb²⁺ 101 Co²⁺96 Fe²⁺ 100 Cu²⁺ 23 Fe³⁺ 102 Ni²⁺ 93 Mn²⁺ 142 Al³⁺ 100 EDTA 24

As evident form the results in Table 7, the enzyme activity was stronglyinhibited by Hg²⁺, Cu²⁺, and EDTA, and it was also inhibited by Ba²⁺ andSr²⁺. It was also found that the enzyme was activated by Ca²⁺ and Mn²⁺.Amino acid analysis on the N-terminal amino acid sequence of the enzymeby “PROTEIN SEQUENCER MODEL 473A”, an apparatus of Applied Biosystems,Inc., Foster City, USA, revealed that the enzyme had a partial aminoacid sequence of SEQ ID NO:1, i.e,tyrosine-valine-serine-serine-leucine-glycine-asparagine-leucine-isoleucinein the N-terminal region. Comparison of the partial amino acid sequencein the N-terminal region with that derived from theα-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporus C9strain in Experiment 5-1 revealed that they were the same and theconsensus N-terminal amino acid sequence, commonly found in theseα-isomaltosylglucosaccharide-forming enzymes, was an amino acid sequenceoftyrosine-valine-serine-serine-leucine-glycine-asparagine-leucine-isoleucineof SEQ ID NO:1 in the N-terminal region. Detailed method for assayingamino acid sequence is not shown in this specification because it isdisclosed in detail in Japanese Patent Application No. 2001-519,441(International Publication No. WO 02/055708), however, theα-isomaltosylglucosaccharide-forming enzyme has an amino acid sequenceof 36-1284 amino acid residues shown in parallel in SEQ ID NO:21similarly as that for the polypeptide, disclosed in the specification ofthe above-identified Japanese Patent Application No. 2001-5441.

EXPERIMENT 8-2 Property of α-isomaltosyl-transferring Enzyme

A purified specimen of α-isomaltosyl-transferring enzyme, obtained bythe method in Experiment 7-3, was subjected to SDS-PAGE using a 7.5%(w/v) of polyacrylamide gel and then determined for molecular weight bycomparing with the dynamics of standard molecular markerselectrophoresed in parallel, commercialized by Japan Bio-RadLaboratories Inc., Tokyo, Japan, revealing that the enzyme had amolecular weight of about 102,000±20,000 daltons.

A fresh preparation of the same purified specimen as used in the abovewas subjected to isoelectrophoresis using a gel containing 2% (w/v)ampholine commercialized by Amersham Corp., Div., AmershamInternational, Arlington Heights, Ill., USA, and then measured for pHsof protein bands and gel to determine the isoelectric point of theenzyme, revealing that the enzyme had an isoelectric point of about5.6±0.5.

The influence of temperature and pH on the activity ofα-isomaltosyl-transferring enzyme was examined in accordance with theassay for its enzyme activity. These results are in FIG. 17 (influenceof temperature) and FIG. 18 (influence of pH). The optimum temperatureof the enzyme was about 50° C. when incubated at pH 6.0 for 30 min. Theoptimum pH of the enzyme was about 5.5 to about 6.0 when incubated at35° C. for 30 min. The thermal stability of the enzyme was determined byincubating the testing enzyme solutions in the form of 20 mM acetatebuffer (pH 6.0) at prescribed temperatures for 60 min, cooling theresulting enzyme solutions with water, and assaying the remaining enzymeactivity of each solution. The pH stability of the enzyme was determinedby keeping the testing enzyme solutions in the form of 50 mM buffershaving prescribed pHs at 4° C. for 24 hours, adjusting the pH of eachsolution to 6.0, and assaying the remaining enzyme activity of eachsolution. These results are respectively in FIG. 19 (thermal stability)and FIG. 20 (pH stability). As a result, the enzyme had thermalstability of up to about 40° C. and pH stability of about 4.5 to about9.0.

The influence of metal ions on the activity ofα-isomaltosyl-transferring enzyme was examined in the presence of 1 mMof each metal-ion according to the assay for its enzyme activity. Theresults are in Table 8.

TABLE 8 Relative activity Metal Relative activity Metal ion (%) ion (%)None 100 Hg²⁺ 2 Zn²⁺ 83 Ba²⁺ 90 Mg²⁺ 91 Sr²⁺ 93 Ca²⁺ 91 Pb²⁺ 74 Co²⁺ 89Fe²⁺ 104 Cu²⁺ 56 Fe³⁺ 88 Ni²⁺ 89 Mn²⁺ 93 Al³⁺ 89 EDTA 98

As evident form the results in Table 8, the enzyme activity was stronglyinhibited by Hg²⁺, and it was also inhibited by Cu²⁺. It was also foundthat the enzyme was not activated by Ca²⁺ and not inhibited by EDTA.

Amino acid analysis on the N-terminal amino acid sequence of the enzymeby “PROTEIN SEQUENCER MODEL 473A”, an apparatus of Applied Biosystems,Inc., Foster City, USA, revealed that the enzyme had a partial aminoacid sequence of SEQ ID NO:3, i.e., isoleucine-asparticacid-glycine-valine-tyrosine-histidine-alanine-proline-tyrosine-glycinein the N-terminal region. Comparison of the partial amino acid sequencein the N-terminal region with that derived from theα-isomaltosyl-transferring enzyme from Bacillus globisporus C9 strain inExperiment 5-2 revealed that they had a consensus amino acid sequence ofisoleucine-asparticacid-glycine-valine-tyrosine-histidine-alanine-proline, as shown in SEQID NO:4 in their N-terminal regions. Detailed method for assaying aminoacid sequence is not shown in this specification because it is disclosedin detail in Japanese Patent Application No. 2000-350142 (InternationalPublication No. WO 02/40659), however, the α-isomaltosyl-transformingenzyme has an amino acid sequence of amino acid residues 30-1093 shownin parallel in SEQ ID NO:22 similarly as that disclosed in thespecification of the above-identified Japanese Patent Application No.2000-350142.

EXPERIMENT 9 Amino Acid Sequence of α-isomaltosylglucosaccharide-formingEnzyme and α-isomaltosyl-transferring Enzyme EXPERIMENT 9-1 InternalPartial Amino Acid Sequence of α-isomaltosylglucosaccharide-formingEnzyme

A part of a purified specimen of α-isomaltosylglucosaccharide-formingenzyme, obtained by the method in Experiment 7-2, was dialyzed against10 mM Tris-HCl buffer (pH 9.0), and the dialyzed solution was dilutedwith a fresh preparation of the same buffer as used in the above to givea concentration of about one milligram per milliliter. One milliliter ofthe dilute as a test sample was admixed with 10 μg of trypsincommercialized by Wako Pure Chemical Industries, Ltd., Tokyo, Japan, andincubated at 30° C. for 22 hours to hydrolyze the enzyme into peptides.To isolate the peptides, the resulting hydrolyzates were subjected toreverse-phase HPLC using “μ-Bondapak C18 column” with a diameter of 2.1mm and a length of 150 mm, a product of Waters Chromatography Div.,MILLIPORE Corp., Milford, USA, at a flow rate of 0.9 ml/min and atambient temperature, and using a liner gradient of acetonitrileincreasing from 8% (v/v) to 40% (v/v) in 0.1% (v/v) trifluoroacetateover 120 min. The peptides eluted from the column were detected bymonitoring the absorbance at a wavelength of 210 nm. Three peptidespecimens named P64 with a retention time of about 64 min, P88 with aretention time of about 88 min, and P99 with a retention time of about99 min, which had been well separated from other peptides, wereseparately collected and dried in vacuo and then dissolved in 200 μl ofa solution of 0.1% (v/v) trifluoroacetate and 50% (v/v) acetonitrile.Each peptide specimen was subjected to a protein sequencer for analyzingamino acid sequence up to eight amino acid residues to obtain amino acidsequences of SEQ ID NOs:5 to 7. The analyzed internal partial amino acidsequences are in Table 9.

TABLE 9 Peptide name Internal partial amino acid sequence P64 asparticacid-alanine-serine-alanine- asparagine-valine-threonine-threonine P88tryptophane-serine-leucine-glycine- phenylalanine-methionine-asparagine-phenylalanine P99 asparagine-tyrosine-threonine-aspartic acid-alanine-tryptophane-methionine-phenylalanine

EXPERIMENT 9-2 Internal Partial Amino Acid Sequence ofα-isomaltosyl-transferring Enzyme

A part of a purified specimen of α-isomaltosyl-transferring enzyme,obtained by the method in Experiment 7-3, was dialyzed against 10 mMTris-HCl buffer (pH 9.0), and the dialyzed solution was diluted with afresh preparation of the same buffer as used in the above to give aconcentration of about one milligram per milliliter. One milliliter ofthe dilute as a test sample was admixed with 10 μg of “LysylEndopeptidase” commercialized by Wako Pure Chemical Industries, Ltd.,Tokyo, Japan, and allowed to react at 30° C. for 22 hours to formpeptides. The resultant mixture was subjected to reverse-phase HPLC toseparate the peptides using “μ-Bondapak C18 column” having a diameter of2.1 mm and a length of 150 mm, a product of Waters Chromatography Div.,MILLIPORE Corp., Milford, USA, at a flow rate of 0.9 ml/min and atambient temperature, and using a liner gradient of acetonitrileincreasing from 8% (v/v) to 40% (v/v) in 0.1% (v/v) trifluoroacetateover 120 min. The peptides eluted from the column were detected bymonitoring the absorbance at a wavelength of 210 nm. Three peptidespecimens named P22 with a retention time of about 22 min, P63 with aretention time of about 63 min, and P71 with a retention time of about71 min, which had been well separated from other peptides, wereseparately collected and dried in vacuo and then dissolved in 200 μl ofa solution of 0.1% (v/v) trifluoroacetate and 50% (v/v) acetonitrile.Each peptide specimen was subjected to a protein sequencer for analyzingamino acid sequence up to eight amino acid residues to obtain amino acidsequences of SEQ ID NOs:8 to 10. The analyzed internal partial aminoacid sequences are in Table 10.

TABLE 10 Peptide name Internal partial amino acid sequence P22glycine-asparagine-glutamic acid-methionine-arginine-asparagine-glutamine-tyrosine P63isoleucine-threonine-threonine-tryptophane- proline-isoleucine-glutamicacid-serine P71 tryptophane-alanine-phenylalanine-glycine-leucine-tryptophane-methionine-serine

EXPERIMENT 10 Production of α-isomaltosylglucosaccharide-forming Enzymefrom Bacillus globisporus N75 Strain

A liquid nutrient culture medium, consisting of 4.0% (w/v) of “PINE-DEX#4”, a partial starch hydrolyzate, 1.8% (w/v) of “ASAHIMEAST”, a yeastextract, 0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodiumphosphate dodecahydrate, 0.05% (w/v) magnesium sulfate heptahydrate, andwater, was placed in 500-ml Erlenmeyer flasks in a volume of 100 mleach, autoclaved at 121° C. for 20 minutes to effect sterilization,cooled, inoculated with a stock culture of Bacillus globisporus N75strain (FERM BP-7591), and incubated at 27° C. for 48 hours under rotaryshaking conditions of 230 rpm for use as a seed culture. About 20 L of afresh preparation of the same nutrient culture medium as used in theabove culture were placed in a 30-L fermentor, sterilized by heating,cooled to 27° C., inoculated with 1% (v/v) of the seed culture, andincubated for about 48 hours while stirring under aeration-agitationconditions at 27° C. and pH 6.0 to 8.0. The resultant culture, havingabout 0.34 unit/ml of an α-isomaltosylglucosaccharide-forming enzymeactivity, about 1.1 units/ml of an α-isomaltosyl-transferring enzymeactivity, and about 0.69 unit/ml of a cyclotetrasaccharide-formingenzyme activity, was centrifuged at 10,000 rpm for 30 min to obtainabout 18 L of a supernatant. Assay for enzyme activity of thesupernatant revealed that it had about 0.33 unit/ml of anα-isomaltosylglucosaccharide-forming enzyme activity, i.e., a totalenzyme activity of about 5,940 units; about 1.1 units/ml of anα-isomaltosyl-transferring enzyme activity, i.e., a total enzymeactivity of about 19,800 units; and about 0.67 unit/ml of acyclotetrasaccharide-forming enzyme activity, i.e., a total enzymeactivity of about 12,100 units.

EXPERIMENT 11 Preparation of Enzyme from Bacillus globisporus N75 Strain

About 18 L of the supernatant obtained in Experiment 10 was salted outin a 60% saturated ammonium sulfate solution and allowed to stand at 4°C. for 24 hours. Then, the salted out sediments were collected bycentrifugation at 10,000 for 30 min, dissolved in 10 mM Tris-HCl buffer(pH 8.3), and dialyzed against a fresh preparation of the same buffer asused in the above to obtain about 450 ml of a crude enzyme solution,revealing to have 4,710 units of α-isomaltosylglucosaccharide-formingenzyme, about 15,700 units of α-isomaltosyl-transferring enzyme, andabout 9,590 units of cyclotetrasaccharide-forming enzyme. The crudeenzyme solution was subjected to ion-exchange chromatography using“SEPABEADS FP-DA13” gel, disclosed in Experiment 4-1. The enzyme wasadsorbed on the gel, while α-isomaltosyl-transferring enzyme was elutedas a non-adsorbed fraction without adsorbing on the gel. When elutedwith a linear gradient increasing from 0 M to 1 M NaCl,α-isomaltosylglucosaccharide-forming enzyme was eluted at aconcentration of about 0.25 M NaCl. Under these conditions, fractionswith an α-isomaltosylglucosaccharide-forming enzyme activity and thosewith an α-isomaltosyl-transferring enzyme were separately fractionatedand collected. Similarly as in the case of Bacillus globisporus C9strain in Experiment 4 and Bacillus globisporus C11 strain in Experiment7, it was revealed that no cyclotetrasaccharide-forming activity wasfound in any of the above fractions collected separately in this columnchromatography, and an enzyme solution, obtained by mixing bothfractions of α-isomaltosylglucosaccharide-forming enzyme and ofα-isomaltosyl-transferring enzyme, showed a cyclotetrasaccharide-formingactivity, and these facts revealed that the activity of formingcyclotetrasaccharide from partial starch hydrolyzates is exerted by thecoaction of α-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme.

The following experiments are methods for separately purifyingα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme:

EXPERIMENT 11-2 Purification of α-isomaltosylglucosaccharide-formingEnzyme

Fractions with α-isomaltosylglucosaccharide-forming enzyme, obtained inExperiment 11-1, were pooled and dialyzed against 10 mM phosphate buffer(pH 7.0) containing 1 M ammonium sulfate, and the dialyzed solution wascentrifuged to remove insoluble impurities and fed to affinitychromatography using 500 ml of “SEPHACRYL HR S-200” gel. The enzyme wasadsorbed on the gel and then eluted therefrom sequentially with a lineargradient decreasing from 1 M to 0 M ammonium sulfate and with a lineargradient increasing from 0 mM to 100 mM maltotetraose. As a result, theα-isomaltosylglucosaccharide-forming enzyme adsorbed on the gel waseluted therefrom at a concentration of about 30 mM maltotetraose,followed by collecting fractions with the enzyme activity. The fractionswere pooled and dialyzed against 10 mM phosphate buffer (pH 7.0)containing 1 M ammonium sulfate, and the dialyzed solution wascentrifuged to remove insoluble impurities. The resulting supernatantwas fed to hydrophobic chromatography using 350 ml of “BUTYL-TOYOPEARL650M”, a gel commercialized by Tosoh Corporation, Tokyo, Japan. Theenzyme was adsorbed on the gel and then eluted with a linear gradientdecreasing from 1 M to 0 M ammonium sulfate, resulting in an elution ofthe enzyme from the gel at a concentration of about 0.3 M ammoniumsulfate and collecting fractions with the enzyme activity. The fractionswere pooled and dialyzed against 10 mM phosphate buffer (pH 7.0)containing 1 M ammonium sulfate, and the dialyzed solution wascentrifuged to remove insoluble impurities and purified on affinitychromatography using 350 ml of “SEPHACRYL HR S-200” gel. The amount ofenzyme activity, specific activity, and yield of theα-isomaltosylglucosaccharide-forming enzyme in each purification stepare in Table 11.

TABLE 11 Specific activity Enzyme* activity of enzyme* YieldPurification step (unit) (unit/mg protein) (%) Culture supernatant 5,9400.10 100 Dialyzed solution after 4,710 0.19 79.3 salting out withammonium sulfate Eluate from ion-exchange 3,200 2.12 53.9 columnchromatography Eluate from affinity 2,210 7.55 37.2 columnchromatography Eluate from hydrophobic 1,720 10.1 29.0 columnchromatography Eluate from affinity 1,320 12.5 22.2 columnchromatography Note: The symbol “*” meansα-isomaltosylglucosaccharide-forming enzyme.

The finally purified α-isomaltosylglucosaccharide-forming enzymespecimen was assayed for purity on gel electrophoresis using a 7.5%(w/v) polyacrylamide gel and detected on the gel as a single proteinband, meaning a high purity enzyme specimen.

EXPERIMENT 11-3 Purification of α-isomaltosyl-transferring Enzyme

Fractions of α-isomaltosyl-transferring enzyme, which had been separatedfrom fractions of α-isomaltosylglucosaccharide-forming enzyme byion-exchange chromatography in Experiment 11-1, were pooled and dialyzedagainst 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate,and the dialyzed solution was centrifuged to remove insolubleimpurities. The resulting supernatant was fed to affinity columnchromatography using 500 ml of “SEPHACRYL HR S-200”, a gelcommercialized by Amersham Corp., Div., Amersham International,Arlington Heights, Ill., USA. The enzyme was adsorbed on the gel andthen eluted therefrom with a linear gradient decreasing from 1 M to 0 Mof ammonium sulfate, resulting in an elution of the enzyme from the gelat a concentration of about 0.3 M ammonium sulfate and collectingfractions with the enzyme activity. The fractions were pooled anddialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammoniumsulfate, and the dialyzed solution was centrifuged to remove insolubleimpurities and purified on hydrophobic chromatography using 380 ml of“BUTYL-TOYOPEARL 650M” gel. The enzyme was adsorbed on the gel and theneluted therefrom with a linear gradient decreasing from 1 M to 0 Mammonium sulfate, resulting in an elution of the enzyme at aconcentration of about 0.3 M ammonium sulfate. The fractions with theenzyme activity were pooled and dialyzed against 10 mM Tris-HCl buffer(pH 8.0), and the dialyzed solution was centrifuged to remove insolubleimpurities. The resulting supernatant was fed to ion-exchange columnchromatography using 380 ml of “SUPER Q-TOYOPEARL 650C” gelcommercialized by Tosoh Corporation, Tokyo, Japan. The enzyme was notadsorbed on the gel and then eluted therefrom as non-adsorbed fractionswhich were then collected and pooled to obtain a finally purified enzymepreparation. The amount of enzyme activity, specific activity, and yieldof the α-isomaltosylglucosaccharide-forming enzyme in each purificationstep are in Table 12.

TABLE 12 Specific activity Enzyme* activity of enzyme* YieldPurification step (unit) (unit/mg protein) (%) Culture supernatant19,000 0.33 100 Dialyzed solution after 15,700 0.64 82.6 salting outwith ammonium sulfate Eluate from ion-exchange 12,400 3.56 65.3 columnchromatography Eluate from affinity 8,320 11.7 43.8 columnchromatography Eluate from hydrophobic 4,830 15.2 25.4 columnchromatography Eluate from ion-exchange 3,850 22.6 20.3 columnchromatography Note: The symbol “*” means α-isomaltosyl-transferringenzyme.

The finally purified α-isomaltosyl-transferring enzyme specimen wasassayed for purity on gel electrophoresis using a 7.5% (w/v)polyacrylamide gel and detected on the gel as a single protein band,meaning a high purity enzyme specimen.

EXPERIMENT 12 Property of α-isomaltosylglucosaccharide-forming Enzymeand α-isomaltosyl-transferring Enzyme EXPERIMENT 12-1 Property ofα-isomaltosylglucosaccharide-forming Enzyme

A purified specimen of α-isomaltosylglucosaccharide-forming enzyme,obtained by the method in Experiment 11-2, was subjected to SDS-PAGEusing a 7.5% (w/v) of polyacrylamide gel and then determined formolecular weight by comparing with the dynamics of standard molecularmarkers electrophoresed in parallel, commercialized by Japan Bio-RadLaboratories Inc., Tokyo, Japan, revealing that the enzyme had amolecular weight of about 136,000±20,000 daltons.

A fresh preparation of the same purified specimen as used in the abovewas subjected to isoelectrophoresis using a gel containing 2% (w/v)ampholine commercialized by Amersham Corp., Div., AmershamInternational, Arlington Heights, Ill., USA, and then measured for pHsof protein bands and gel to determine the isoelectric point of theenzyme, revealing that the enzyme had an isoelectric point of about7.3±0.5.

The influence of temperature and pH on the activity ofα-isomaltosylglucosaccharide-forming enzyme was examined in accordancewith the assay for its enzyme activity, where the influence oftemperature was examined in the presence or the absence of 1 mM Ca²⁺.These results are in FIG. 21 (influence of temperature) and FIG. 22(influence of pH). The optimum temperature of the enzyme was about 50°C. and about 55° C. when incubated at pH 6.0 for 60 min in the absenceof and in the presence of 1 mM Ca²⁺, respectively. The optimum pH of theenzyme was about 6.0 when incubated at 35° C. for 60 min. The thermalstability of the enzyme was determined by incubating the testing enzymesolutions in the form of 20 mM acetate buffer (pH 6.0) at prescribedtemperatures for 60 min in the absence of and in the presence of 1 mMCa²⁺, cooling the resulting enzyme solutions with water, and assayingthe remaining enzyme activity of each solution. The pH stability of theenzyme was determined by keeping the testing enzyme solutions in theform of 50 mM buffers having prescribed pHs at 4° C. for 24 hours,adjusting the pH of each solution to 6.0, and assaying the remainingenzyme activity of each solution. These results are respectively in FIG.23 (thermal stability) and FIG. 24 (pH stability). As a result, theenzyme had thermal stability of up to about 45° C. and about 50° C. inthe absence of and in the presence of 1 mM Ca²⁺, respectively, and hadpH stability of about 5.0 to about 9.0.

The influence of metal ions on the activity ofα-isomaltosylglucosaccharide-forming enzyme was examined in the presenceof 1 mM of each metal-ion according to the assay for its enzymeactivity. The results are in Table 13.

TABLE 13 Relative Relative Metal ion activity (%) Metal ion activity (%)None 100 Hg²⁺ 1 Zn²⁺ 82 Ba²⁺ 84 Mg²⁺ 96 Sr²⁺ 85 Ca²⁺ 108 Pb²⁺ 86 Co²⁺ 93Fe²⁺ 82 Cu²⁺ 7 Fe³⁺ 93 Ni²⁺ 93 Mn²⁺ 120 Al³⁺ 98 EDTA 35

As evident form the results in Table 13, the enzyme activity wasstrongly inhibited by Hg²⁺, Cu²⁺, and EDTA. It was also found that theenzyme was activated by Ca²⁺ and Mn²⁺. Amino acid analysis on theN-terminal amino acid sequence of the enzyme by “PROTEIN SEQUENCER MODEL473A”, an apparatus of Applied Biosystems, Inc., Foster City, USA,revealed that the enzyme had a partial amino acid sequence of SEQ IDNO:11, i.e.,histidine-valine-serine-alanine-leucine-glycine-asparagine-leucine-leucinein the N-terminal region. Comparison of the above partial amino acidsequence in the N-terminal region with that derived from theα-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporusC11 strain in Experiment 8-1 revealed that they had a relatively highhomology but differed in the amino acid residues 1, 4 and 9 in each oftheir partial amino acid sequences in their N-terminal regions. Detailedmethod for assaying amino acid sequence is not shown in thisspecification because it is disclosed in detail in Japanese PatentApplication No. 2001-5441 (International Publication No. WO02/055708),however, the α-isomaltosylglucosaccharide-forming enzyme has an aminoacid sequence of amino acid residues 36-1286 shown in parallel in SEQ IDNO:23 similarly as that disclosed in the specification of theabove-identified Japanese Patent Application No. 2001-5441.

EXPERIMENT 12-2 Property of α-isomaltosyl-transferring Enzyme

A purified specimen of α-isomaltosyl-transferring enzyme, obtained bythe method in Experiment 11-3, was subjected to SDS-PAGE using a 7.5%(w/v) of polyacrylamide gel and then determined for molecular weight bycomparing with the dynamics of standard molecular markerselectrophoresed in parallel, commercialized by Japan Bio-RadLaboratories Inc., Tokyo, Japan, revealing that the enzyme had amolecular weight of about 112,000±20,000 daltons.

A fresh preparation of the same purified specimen as used in the abovewas subjected to isoelectrophoresis using a gel containing 2% (w/v)ampholine commercialized by Amersham Corp., Div., AmershamInternational, Arlington Heights, Ill., USA, and then measured for pHsof protein bands and gel to determine the isoelectric point of theenzyme, revealing that the enzyme had an isoelectric point of about7.8±0.5.

The influence of temperature and pH on the activity ofα-isomaltosyl-transferring enzyme was examined in accordance with theassay for its enzyme activity. These results are in FIG. 25 (influenceof temperature) and FIG. 26 (influence of pH). The optimum temperatureof the enzyme was about 50° C. when incubated at pH 6.0 for 30 min. Theoptimum pH of the enzyme was about 6.0 when incubated at 35° C. for 30min. The thermal stability of the enzyme was determined by incubatingthe testing enzyme solutions in the form of 20 mM acetate buffer (pH6.0) at prescribed temperatures for 60 min, cooling the resulting enzymesolutions with water, and assaying the remaining enzyme activity of eachsolution. The pH stability of the enzyme was determined by keeping thetesting enzyme solutions in the from of 50 mM buffers having prescribedpHs at 4° C. for 24 hours, adjusting the pH of each solution to 6.0, andassaying the remaining enzyme activity of each solution. These resultsare respectively in FIG. 27 (thermal stability) and FIG. 28 (pHstability). As a result, the enzyme had thermal stability of up to about45° C. and had pH stability of about 4.5 to about 10.0. The influence ofmetal ions on the activity of α-isomaltosyl-transferring enzyme wasexamined in the presence of 1 mM of each metal-ion according to theassay for its enzyme activity. The results are in Table 14.

TABLE 14 Relative activity Metal Relative activity Metal ion (%) ion (%)None 100 Hg²⁺ 0.5 Zn²⁺ 75 Ba²⁺ 102 Mg²⁺ 95 Sr²⁺ 91 Ca²⁺ 100 Pb²⁺ 69 Co²⁺92 Fe²⁺ 97 Cu²⁺ 15 Fe³⁺ 90 Ni²⁺ 91 Mn²⁺ 101 Al³⁺ 94 EDTA 92

As evident form the results in Table 14, the enzyme activity wasstrongly inhibited by Hg²⁺ and also inhibited by Cu²⁺. It was also foundthat the enzyme was not activated by Ca²⁺ and not inhibited by EDTA.

Amino acid analysis on the N-terminal amino acid sequence of the enzymeby “PROTEIN SEQUENCER MODEL 473A”, an apparatus of Applied Biosystems,Inc., Foster City, USA, revealed that the enzyme had a partial aminoacid sequence of SEQ ID NO:3, i.e., isoleucine-asparticacid-glycine-valine-tyrosine-histidine-alanine-proline-tyrosine-glycineat the N-terminal region. Comparison of the above partial amino acidsequence at the N-terminal region with that derived from theα-isomaltosyl-transferring enzymes from Bacillus globisporus C9 strainin Experiment 5-2 and from Bacillus globisporus C11 strain in Experiment8-2 revealed that they had a consensus amino acid sequence ofisoleucine-asparticacid-glycine-valine-tyrosine-histidine-alanine-proline, as shown in SEQID NO:4 in their N-terminal regions. Detailed method for assaying aminoacid sequence is not shown in this specification because it is disclosedin detail in PCT/JP01/04276 (International Publication No. WO 01/90338),however, the α-isomaltosyl-transferring enzyme obtained in Experiment11-3 has an amino acid sequence of amino acid residues 30-1093 shown inparallel in SEQ ID NO:24 similarly as the polypeptide disclosed in thespecification of PCT/JP01/04276.

EXPERIMENT 13 Internal Amino Acid Sequence ofα-isomaltosylglucosaccharide-forming Enzyme andα-isomaltosyl-transferring Enzyme EXPERIMENT 13-1 Internal Partial AminoAcid Sequence of α-isomaltosylglucosaccharide-forming Enzyme

A part of a purified specimen of α-isomaltosylglucosaccharide-formingenzyme, obtained by the method in Experiment 11-2, was dialyzed against10 mM Tris-HCl buffer (pH 9.0), and the dialyzed solution was dilutedwith a fresh preparation of the same buffer as used in the above to givea concentration of about one milligram per milliliter. One milliliter ofthe dilute as a test sample was admixed with 20 μg of “LysylEndopeptidase” commercialized by Wako Pure Chemical Industries, Ltd.,Tokyo, Japan, and allowed to react at 30° C. for 24 hours to formpeptides. The resultant mixture was subjected to reverse-phase HPLC toseparate the peptides using “μ-Bondasphere C18 column” having a diameterof 3.9 mm and a length of 150 mm, a product of Waters ChromatographyDiv., MILLIPORE Corp., Milford, USA, at a flow rate of 0.9 ml/min and atambient temperature, and using a liner gradient of acetonitrileincreasing from 8% (v/v) to 36% (v/v) in 0.1% (v/v) trifluoroacetateover 120 min. The peptides eluted from the column were detected bymonitoring the absorbance at a wavelength of 210 nm. Three peptidespecimens named PN59 with a retention time of about 59 min, PN67 with aretention time of about 67 min, and PN87 with a retention time of about87 min, which had been well separated from other peptides, wereseparately collected and dried in vacuo and then dissolved in 200 μl ofa solution of 0.1% (v/v) trifluoroacetate and 50% (v/v) acetonitrile.Each peptide specimen was subjected to a protein sequencer for analyzingamino acid sequence up to eight amino acid residues to obtain amino acidsequences of SEQ ID NOs:12 to 14. The analyzed internal partial aminoacid sequences are in Table 15.

TABLE 15 Peptide name Internal partial amino acid sequence PN59 asparticacid-phenylalanine-serine- asparagine-asparagine-proline-threonine-valine PN67 tyrosine-threonine-valine-asparagine-alanine-proline-alanine-alanine PN87 tyrosine-glutamicacid-alanine-glutamic acid-serine-alanine-glutamic acid-leucine

EXPERIMENT 13-2 Internal Amino Acid Sequence ofα-isomaltosyl-transferring Enzyme

A part of a purified specimen of α-isomaltosyl-transferring enzyme,obtained by the method in Experiment 11-3, was dialyzed against 10 mMTris-HCl buffer (pH 9.0), and the dialyzed solution was diluted with afresh preparation of the same buffer as used in the above to give aconcentration of about one milligram per milliliter. One milliliter ofthe dilute as a test sample was admixed with 20 μg of “LysylEndopeptidase” commercialized by Wako Pure Chemical Industries, Ltd.,Tokyo, Japan, and allowed to react at 30° C. for 24 hours to formpeptides. The resultant mixture was subjected to reverse-phase HPLC toseparate the peptides using “μ-Bondasphere C18 column” having a diameterof 3.9 mm and a length of 150 mm, a product of Waters ChromatographyDiv., MILLIPORE Corp., Milford, USA, at a flow rate of 0.9 ml/min and atambient temperature, and using a liner gradient of acetonitrileincreasing from 4% (v/v) to 42.4% (v/v) in 0.1% (v/v) trifluoroacetateover 90 min. The peptides eluted from the column were detected bymonitoring the absorbance at a wavelength of 210 nm. Three peptidespecimens named PN21 with a retention time of about 21 min, PN38 with aretention time of about 38 min, and PN69 with a retention time of about69 min which had been well separated from other peptides, wereseparately collected and dried in vacuo and then dissolved in 200 μl ofa solution of 0.1% (v/v) trifluoroacetate and 50% (v/v) acetonitrile.Each peptide specimen was subjected to a protein sequencer for analyzingamino acid sequence up to eight amino acid residues, but up to six aminoacids residues for PN21, to obtain amino acid sequences of SEQ ID NOs:15 to 17. The analyzed internal partial amino acid sequences are inTable 16.

TABLE 16 Peptide name Internal partial amino acid sequence PN21asparagine-tryptophane-tryptophane- methionine-serine-lysine PN38threonine-aspartic acid-glycine-glycine- glutamicacid-methionine-valine-tryptophane PN69asparagine-isoleucine-tyrosine-leucine-proline-glutamine-glycine-aspartic acid

EXPERIMENT 14 Production of α-isomaltosylglucosaccharide-forming Enzymefrom Arthrobacter globiformis A19 Strain

A liquid nutrient culture medium, consisting of 4.0% (w/v) of “PINE-DEX#4”, a partial starch hydrolyzate, 1.8% (w/v) of “ASAHIMEAST”, a yeastextract, 0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodiumphosphate dodecahydrate, 0.05% (w/v) magnesium sulfate heptahydrate, andwater, was placed in 500-ml Erlenmeyer flasks in a volume of 100 mleach, autoclaved at 121° C. for 20 minutes to effect sterilization,cooled, inoculated with a stock culture of Arthrobacter globiformis A19strain (FERM BP-7590), and incubated at 27° C. for 48 hours under rotaryshaking conditions of 230 rpm for use as a seed culture. About 20 L of afresh preparation of the same nutrient culture medium as used in theabove culture were placed in a 30-L fermentor, sterilized by heating,cooled to 27° C., inoculated with 1% (v/v) of the seed culture, andincubated for about 48 hours while stirring under aeration-agitationconditions at 27° C. and pH 6.0 to 9.0. The resultant culture, havingabout 1.1 units/ml of an α-isomaltosylglucosaccharide-forming enzymeactivity, about 1.7 units/ml of an α-isomaltosyl-transferring enzymeactivity, and about 0.35 unit/ml of a cyclotetrasaccharide-formingenzyme activity, was centrifuged at 10,000 rpm for 30 min to obtainabout 18 L of a supernatant. Measurement of the supernatant revealedthat it had about 1.06 units/ml of anα-isomaltosylglucosaccharide-forming enzyme activity, i.e., a totalenzyme activity of about 19,100 units; about 1.6 units/ml of anα-isomaltosyl-transferring enzyme activity, i.e., a total enzymeactivity of about 28,800 units; and about 0.27 unit/ml of acyclotetrasaccharide-forming enzyme activity, i.e., a total enzymeactivity of about 4,860 units. The activity of theα-isomaltosylglucosaccharide-forming enzyme from Arthrobacterglobiformis A19 strain was similarly assayed as the method in Experiment3 except for using 100 mM glycine-NaOH buffer (pH 8.4) as a buffer forsubstrate.

EXPERIMENT 15 Preparation of Enzyme from Arthrobacter globiformis A19Strain EXPERIMENT 15-1 Purification of Enzyme from Arthrobacterglobiformis A19 Strain

About 18 L of the supernatant, obtained in Experiment 14, was salted outin a 60% saturated ammonium sulfate solution and allowed to stand at 4°C. for 24 hours. Then, the salted out sediments were collected bycentrifugation at 10,000 for 30 min, dissolved in 10 mM phosphate buffer(pH 7.0), dialyzed against a fresh preparation of the same buffer asused in the above to obtain about 850 ml of a crude enzyme solution. Thecrude enzyme solution was revealed to have 8,210 units ofα-isomaltosylglucosaccharide-forming enzyme, about 15,700 units ofα-isomaltosyl-transferring enzyme, and about 2,090 units ofcyclotetrasaccharide-forming enzyme, followed by subjecting it toion-exchange chromatography using 380 ml of “DEAE-TOYOPEARL 650S” gel.When eluted with a linear gradient increasing from 0 M to 0.5 M NaCl,α-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme were separately eluted from the gel,the former was eluted at a concentration of about 0.2 M NaCl, while thelatter was eluted at a concentration of about 0.3 M NaCl. Under theseconditions, fractions with an α-isomaltosylglucosaccharide-formingenzyme activity and those with an α-isomaltosyl-transferring enzymeactivity were separately fractionated and collected. Since the factsthat no cyclotetrasaccharide-forming activity was found in any fractionobtained in this column chromatography, and an enzyme solution, obtainedby mixing the fractions of α-isomaltosylglucosaccharide-forming enzymeand of α-isomaltosyl-transferring enzyme, showed acyclotetrasaccharide-forming activity, it was revealed that the activityof forming cyclotetrasaccharide from partial starch hydrolyzates isexerted by the coaction of α-isomaltosylglucosaccharide-forming enzymeand α-isomaltosyl-transferring enzyme.

The following experiments describe a method for separately purifyingα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme:

EXPERIMENT 15-2 Purification of α-isomaltosylglucosaccharide-formingEnzyme

Fractions with α-isomaltosylglucosaccharide-forming enzyme, obtained inExperiment 15-1, were pooled and dialyzed against 10 mM phosphate buffer(pH 7.0) containing 1 M ammonium sulfate, and the dialyzed solution wascentrifuged to remove insoluble impurities and fed to affinitychromatography using 500 ml of “SEPHACRYL HR S-200” gel. The enzyme wasadsorbed on the gel and then eluted therefrom with a linear gradientdecreasing from 1 M to 0 M ammonium sulfate. As a result, theα-isomaltosylglucosaccharide-forming enzyme adsorbed on the gel waseluted therefrom at a concentration of about 0.2 M ammonium sulfate,followed by collecting fractions with the enzyme activity and poolingthem for use as a finally purified specimen. The amount of enzymeactivity, specific activity, and yield ofα-isomaltosylglucosaccharide-forming enzyme in each purification stepare in Table 17.

TABLE 17 Specific activity Enzyme* activity of enzyme* YieldPurification step (unit) (unit/mg protein) (%) Culture supernatant19,100 0.11 100 Dialyzed solution after 8,210 0.48 43.0 salting out withammonium sulfate Eluate from ion-exchange 6,890 4.18 36.1 columnchromatography Eluate from affinity 5,220 35.1 27.3 columnchromatography Note: The symbol “*” meansα-isomaltosylglucosaccharide-forming enzyme.

The finally purified α-isomaltosylglucosaccharide-forming enzymespecimen was assayed for purity on gel electrophoresis using a 7.5%(w/v) polyacrylamide gel and detected on the gel as a single proteinband, meaning a high purity enzyme specimen.

EXPERIMENT 15-3 Purification of α-isomaltosyl-transferring Enzyme

Fractions of α-isomaltosyl-transferring enzyme, which had been separatedfrom fractions of α-isomaltosylglucosaccharide-forming enzyme byion-exchange chromatography in Experiment 15-1, were pooled and dialyzedagainst 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate,and the dialyzed solution was centrifuged to remove insolubleimpurities. The resulting supernatant was fed to affinity columnchromatography using 500 ml of “SEPHACRYL HR S-200” gel, a gelcommercialized by Amersham Corp., Div., Amersham International,Arlington Heights, Ill., USA. The enzyme was adsorbed on the gel andthen eluted therefrom with a linear gradient decreasing from 1 M to 0 Mof ammonium sulfate, resulting in an elution of the enzyme from the gelat a concentration of about 0 M ammonium sulfate and collecting theresulting fractions with the enzyme activity for a partially purifiedspecimen. The amount of enzyme activity, specific activity, and yield ofα-isomaltosyl-transferring enzyme in each purification step are in Table18.

TABLE 18 Specific activity Enzyme* activity of enzyme* YieldPurification step (unit) (unit/mg protein) (%) Culture supernatant28,800 0.18 100 Dialyzed solution after 15,700 0.97 54.5 salting outwith ammonium sulfate Eluate from ion-exchange 7,130 4.01 24.8 columnchromatography Eluate from affinity 1,440 12.1 5.0 column chromatographyNote: The symbol “*” means α-isomaltosyl-transferring enzyme.

The partially purified α-isomaltosyl-transferring enzyme specimen wasassayed for purity on gel electrophoresis using a 7.5% (w/v)polyacrylamide gel and detected on the gel as a single protein band,meaning a high purity enzyme specimen.

EXPERIMENT 16 Property of α-isomaltosylglucosaccharide-forming Enzymeand α-isomaltosyl-transferring Enzyme EXPERIMENT 16-1 Property ofα-isomaltosylglucosaccharide-forming Enzyme

A purified specimen of α-isomaltosylglucosaccharide-forming enzyme,obtained by the method in Experiment 15-2, was subjected to SDS-PAGEusing a 7.5% (w/v) of polyacrylamide gel and then determined formolecular weight by comparing with the dynamics of standard molecularmarkers electrophoresed in parallel, commercialized by Japan Bio-RadLaboratories Inc., Tokyo, Japan, revealing that the enzyme had amolecular weight of about 94,000±20,000 daltons.

A portion of the above purified specimen was subjected toisoelectrophoresis using a gel containing 2% (w/v) ampholinecommercialized by Amersham Corp., Div., Amersham International,Arlington Heights, Ill., USA, and then measured for pHs of protein bandsand gel to determine the isoelectric point of the enzyme, revealing thatthe enzyme had an isoelectric point of about 4.3±0.5.

The influence of temperature and pH on the activity ofα-isomaltosylglucosaccharide-forming enzyme was examined in accordancewith the assay for its enzyme activity. The influence of temperature wasdetermined in the presence of or the absence of 1 mM Ca²⁺. These resultsare in FIG. 29 (influence of temperature) and FIG. 30 (influence of pH).The optimum temperature of the enzyme was about 60° C. and about 65° C.when incubated at pH 8.4 for 60 min in the absence of and in thepresence of 1 mM Ca²⁺, respectively. The optimum pH of the enzyme wasabout 8.4 when incubated at 35° C. for 60 min. The thermal stability ofthe enzyme was determined by incubating the testing enzyme solutions inthe form of 20 mM glycine-NaOH buffer (pH 8.0) at prescribedtemperatures for 60 min in the absence of or the presence of 1 mM Ca²⁺,cooling the resulting enzyme solutions with water, and assaying theremaining enzyme activity of each solution. The pH stability of theenzyme was determined by keeping the testing enzyme in 50 mM buffershaving prescribed pHs at 4° C. for 24 hours, adjusting the pH of eachsolution to 8.0, and assaying the remaining enzyme activity of eachsolution. These results are respectively in FIG. 31 (thermal stability)and FIG. 32 (pH stability). As a result, the enzyme had thermalstability of up to about 55° C. and about 60° C. in the absence of andin the presence of 1 mM Ca²⁺, respectively, and had pH stability ofabout 5.0 to about 9.0.

The influence of metal ions on the activity ofα-isomaltosyl-transferring enzyme was examined in the presence of 1 mMof each metal-ion according to the assay for its enzyme activity. Theresults are in Table 19.

TABLE 19 Relative activity Metal Relative activity Metal ion (%) ion (%)None 100 Hg²⁺ 0 Zn²⁺ 56 Ba²⁺ 99 Mg²⁺ 97 Sr²⁺ 102 Ca²⁺ 106 Pb²⁺ 43 Co²⁺93 Fe²⁺ 36 Cu²⁺ 0 Fe³⁺ 35 Ni²⁺ 46 Mn²⁺ 98 Al³⁺ 37 EDTA 2

As evident form the results in Table 19, it was revealed that the enzymeactivity was strongly inhibited by Hg²⁺, Cu²⁺, and EDTA. Amino acidanalysis on the N-terminal amino acid sequence of the enzyme by “PROTEINSEQUENCER MODEL 473A”, an apparatus of Applied Biosystems, Inc., FosterCity, USA, revealed that the enzyme had a partial amino acid sequence ofSEQ ID NO:18, i.e.,alanine-proline-leucine-glycine-valine-glutamine-arginine-alanine-glutamine-phenylalanine-glutamine-serine-glycinein the N-terminal region. Detailed method for assaying amino acidsequence is not shown in this specification because it is disclosed indetail in Japanese Patent Application No. 2001-5441 (InternationalPublication No. WO 02/055708), however, theα-isomaltosylglucosaccharide-forming enzyme has an amino acid sequenceof amino acid residues 37-965 shown in parallel in SEQ ID NO:25similarly as the polypeptide disclosed in the specification of the aboveJapanese Patent Application No. 2001-5441.

EXPERIMENT 16-2 Property of α-isomaltosyl-transferring Enzyme

A purified specimen of α-isomaltosyl-transferring enzyme, obtained bythe method in Experiment 15-3, was subjected to SDS-PAGE using a 7.5%(w/v) of polyacrylamide gel and then determined for molecular weight bycomparing with the dynamics of standard molecular markerselectrophoresed in parallel, commercialized by Japan Bio-RadLaboratories Inc., Tokyo, Japan, revealing that the enzyme had amolecular weight of about 113,000±20,000 daltons.

A portion of the above purified specimen was subjected toisoelectrophoresis using a gel containing 2% (w/v) ampholinecommercialized by Amersham Corp., Div., Amersham International,Arlington Heights, Ill., USA, and then measured for pHs of protein bandsand gel to determine the isoelectric point of the enzyme, revealing thatthe enzyme had an isoelectric point of about 4.2±0.5.

The influence of temperature and pH on the above enzyme was examined inaccordance with the assay for its enzyme activity. These results are inFIG. 33 (influence of temperature) and FIG. 34 (influence of pH). Theoptimum temperature of the enzyme was about 50° C. when incubated at pH6.0 for 30 min. The optimum pH of the enzyme was about 6.5 whenincubated at 35° C. for 30 min. The thermal stability of the enzyme wasdetermined by incubating the testing enzyme solutions in the form of 20mM acetate buffer (pH 6.0) at prescribed temperatures for 60 min,cooling the resulting enzyme solutions with water, and assaying theremaining enzyme activity of each solution. The pH stability of theenzyme was determined by keeping the testing enzyme solutions in theform of 50 mM buffers having prescribed pHs at 4° C. for 24 hours,adjusting the pH of each solution to 6.0, and assaying the remainingenzyme activity of each solution. These results are respectively in FIG.35 (thermal stability) and FIG. 36 (pH stability). As a result, theenzyme had thermal stability of up to about 45° C. and pH stability ofabout 4.5 to about 9.0. Amino acid analysis on the N-terminal amino acidsequence of the enzyme by “PROTEIN SEQUENCER MODEL 473A”, an apparatusof Applied Biosystems, Inc., Foster City, USA, revealed that the enzymehad a partial amino acid sequence of SEQ ID NO:19, i.e.,asparagine-threonine-leucine-asparticacid-glycine-valine-tryptophane-histidine-asparagine-proline-tyrosine-glycine-alanine-asparticacid-glutamic acid-leucine-tyrosine-alanine-threonine-glutamine in theN-terminal region.

EXPERIMENT 16-3 Total Amino Acid Sequence of α-isomaltosyl-transferringEnzyme

According to the method in Japanese Patent Application No. 2001-5441(International Publication No. WO 02/055708), chromosomal DNAs (cDNAs)were extracted from Arthrobacter globiformis A19 strain and purified.The purified cDNAs were hydrolyzed with a restriction enzyme, Not I, toobtain DNA fragments. While, “Bluescript II SK(+)”, a plasmid vectorcommercialized by Stratagene Cloning Systems, California, USA, wascompletely cleaved with a restriction enzyme, Not I, and the resultingcleaved plasmid vector and the above DNA fragments using “DNA LigationKit” commercialized by Takara Shuzo Co., Ltd., Tokyo, Japan, to obtain arecombinant DNA. “Epicurian Coli XL2-Blue”, commercialized by StratageneCloning Systems, California, USA, was transformed with the recombinantDNA to obtain a gene library. An oligonucleotide, represented by5′-AAYACNCTNGAYGGNGTNTGGCAYAAYCCNTAYGGNGCNGAYGARCTNTGGAC-3′, waschemically synthesized based on the amino acid sequence of amino acidresidues 1-18 in SEQ ID NO:19, which had been revealed by the method inExperiment 16-2; and labeled with [γ-³²P]ATP and T4 polynucleotidekinase to obtain a probe. In accordance with the method in JapanesePatent Application No. 2001-5441 (International Publication No. WO02/055708), the above gene library and the probe were subjected to thecolony hybridization method, followed by selecting a transformant thatstrongly hybridized with the probe. The transformant was named “AGA4”.According to conventional manner, a recombinant DNA was prepared fromthe transformant and analyzed for nucleotide sequence by conventionaldideoxy method, revealing that the recombinant DNA thus obtainedcomprised the DNA of SEQ ID NO:26 consisting of 6153 base pairs, derivedfrom Arthrobacter globiformis A19 strain. As shown in FIG. 37, in therecombinant DNA, the above DNA was linked to the downstream of therecognition site of Not I. When an amino acid sequence estimable fromthe above nucleotide sequence, which is shown in parallel in SEQ IDNO:26, was compared with the N-terminal amino acid sequence of theα-isomaltosyl-transferring enzyme that was confirmed by the method inExperiment 16-2, the amino acid sequence of SEQ ID NO:19 was completelycoincided with the amino acid residues 50-69 shown in parallel in SEQ IDNO:26. Since the nucleotide sequence of nucleotide residues 4644-4646 inSEQ ID NO:26 encodes the termination codon (5′-TGA-3′), the C-terminusof α-isomaltosyl-transferring enzyme was revealed to be arginine,corresponding to amino acid residue 1121, shown in parallel in SEQ IDNO:26, which positions just before the termination codon. These resultsshow that the α-isomaltosyl-transferring enzyme obtained in Experiment15-3 comprises the amino acid residues 50-1121 shown in parallel in SEQID NO:26 and is encoded by a DNA comprising the nucleotide residues1428-4643 shown in parallel in SEQ ID NO:26. A sequence of amino acidresidues 1-49 shown in parallel in SEQ ID NO:26 was estimated to be anamino acid sequence of secretory signal for the polypeptide. These datarevealed that the precursor peptide of the polypeptide before secretioncomprises the amino acid sequence shown in parallel in SEQ ID NO:26 andis encoded by the nucleotide sequence shown in parallel in SEQ ID NO:26.Based on these, the recombinant DNA with its confirmed nucleotidesequence was named “pAGA4”.

EXPERIMENT 17 Production of α-isomaltosyl-transferring Enzyme fromArthrobacter ramosus S1 Strain

A liquid nutrient culture medium, consisting of 4.0% (w/v) of “PINE-DEX#4”, a partial starch hydrolyzate, 1.8% (w/v) of “ASAHIMEAST”, a yeastextract, 0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodiumphosphate dodecahydrate, 0.05% (w/v) magnesium sulfate heptahydrate, andwater, was placed in 500-ml Erlenmeyer flasks in a volume of 100 mleach, autoclaved at 121° C. for 20 min to effect sterilization, cooled,inoculated with a stock culture of Arthrobacter ramosus S1 strain (FERMBP-7592), and incubated at 27° C. for 48 hours under rotary shakingconditions of 230 rpm for use as a seed culture. About 20 L of a freshpreparation of the same nutrient culture medium as used in the aboveculture were placed in a 30-L fermentor, sterilized by heating, cooledto 27° C., inoculated with 1% (v/v) of the seed culture, and incubatedfor about 48 hours while stirring under aeration-agitation conditions at27° C. and pH 6.0 to 8.0. The resultant culture, having about 0.45unit/ml of an α-isomaltosyl-transferring activity, was centrifuged at10,000 rpm for 30 min to obtain about 18 L of a supernatant having about0.44 unit/ml of an α-isomaltosyl-transferring enzyme activity and atotal enzyme activity of about 7,920 units.

EXPERIMENT 18 Purification of α-isomaltosyl-transferring Enzyme fromArthrobacter ramosus S1 Strain

About 18 L of a supernatant obtained in Experiment 17 were salted out inan 80% (w/v) ammonium sulfate solution at 4° C. for 24 hours, and theresulting sediments were collected by centrifugation at 10,000 rpm for30 min and dialyzed against 10 mM phosphate buffer (pH 7.0) to obtainabout 380 ml of a crude enzyme solution having 6,000 units ofα-isomaltosyl-transferring enzyme. The crude enzyme solution wasdialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammoniumsulfate, and the dialyzed solution was centrifuged to remove insolubleimpurities. The resulting supernatant was fed to affinity columnchromatography using 500 ml of “SEPHACRYL HR S-200” gel. The enzyme wasadsorbed on the gel and then eluted sequentially with a linear gradientdecreasing from 1 M to 0 M of ammonium sulfate and with a lineargradient increasing from 0% (w/v) to 5% (w/v) maltotetraose, resultingin an elution of the enzyme from the gel at a concentration of about 2%(w/v) maltotetraose and collecting fractions with the enzyme activity.The fractions were pooled and dialyzed against 10 mM phosphate buffer(pH 7.0) containing 1 M ammonium sulfate, and the dialyzed solution wascentrifuged to remove insoluble impurities. The supernatant thusobtained was fed to hydrophobic column chromatography using 380 ml of“BUTYL-TOYOPEARL 650M” gel. When eluted with a linear gradientdecreasing from 1 M to 0 M ammonium sulfate, theα-isomaltosyl-transferring enzyme adsorbed on the gel was elutedtherefrom at about 0.3 M ammonium sulfate, followed by collectingfractions with the enzyme activity for a purified enzyme specimen. Theamount of enzyme activity, specific activity, and yield of theα-isomaltosyl-transferring enzyme in each purification step are in Table20.

TABLE 20 Specific activity Enzyme* activity of enzyme* YieldPurification step (unit) (unit/mg protein) (%) Culture supernatant 7,9200.47 100 Dialyzed solution after 6,000 3.36 75.8 salting out withammonium sulfate Eluate from affinity 5,270 29.9 66.5 columnchromatography Eluate from hydrophobic 4,430 31.1 55.9 columnchromatography Note: The symbol “*” means α-isomaltosyl-transferringenzyme.

The purified α-isomaltosyl-transferring enzyme specimen obtained in thisexperiment was assayed for purity on gel electrophoresis using a 7.5%(w/v) polyacrylamide gel and detected on the gel as a single proteinband, meaning a high purity enzyme specimen.

EXPERIMENT 19 Property of α-Isomaltosyl-transferring Enzyme

A purified specimen of α-isomaltosyl-transferring enzyme, obtained bythe method in Experiment 18, was subjected to SDS-PAGE using a 7.5%(w/v) of polyacrylamide gel and then determined for molecular weight bycomparing with the dynamics of standard molecular markerselectrophoresed in parallel, commercialized by Japan Bio-RadLaboratories Inc., Tokyo, Japan, revealing that the enzyme had amolecular weight of about 116,000±20,000 daltons.

A portion of the above purified specimen was subjected toisoelectrophoresis using a gel containing 2% (w/v) ampholinecommercialized by Amersham Corp., Div., Amersham International,Arlington Heights, Ill., USA, and then measured for pHs of protein bandsand gel to determine the isoelectric point of the enzyme, revealing thatthe enzyme had an isoelectric point of about 4.2±0.5.

The influence of temperature and pH on the activity ofα-isomaltosyl-transferring enzyme was examined in accordance with theassay for its enzyme activity. These results are in FIG. 38 (influenceof temperature) and FIG. 39 (influence of pH). The optimum temperatureof the enzyme was about 50° C. when incubated at pH 6.0 for 30 min. Theoptimum pH of the enzyme was about 6.0 when incubated at 35° C. for 30min. The thermal stability of the enzyme was determined by incubatingthe testing enzyme solutions in the form of 20 mM acetate buffers (pH6.0) at prescribed temperatures for 60 min, cooling the resulting enzymesolutions with water, and assaying the remaining enzyme activity of eachsolution. The pH stability of the enzyme was determined by keeping thetesting enzyme solutions in the from of 50 mM buffers having prescribedpHs at 4° C. for 24 hours, adjusting the pH of each solution to 6.0, andassaying the remaining enzyme activity of each solution. These resultsare respectively in FIG. 40 (thermal stability) and FIG. 41 (pHstability). As evident from these figures, the enzyme had thermalstability of up to about 45° C. and had pH stability of about 3.6 toabout 9.0.

The influence of metal ions on the activity ofα-isomaltosyl-transferring enzyme was examined in the presence of 1 mMof each metal-ion according to the assay for its enzyme activity. Theresults are in Table 21.

TABLE 21 Metal Relative activity Metal Relative activity ion (%) ion (%)None 100 Hg²⁺ 0.1 Zn²⁺ 78 Ba²⁺ 97 Mg²⁺ 99 Sr²⁺ 101 Ca²⁺ 103 Pb²⁺ 85 Co²⁺91 Fe²⁺ 105 Cu²⁺ 2 Fe³⁺ 75 Ni²⁺ 87 Mn²⁺ 98 Al³⁺ 93 EDTA 91

As evident form the results in Table 21, it was revealed that the enzymeactivity was strongly inhibited by Hg²⁺ and also inhibited by Cu²⁺. Itwas also revealed that the enzyme was neither activated by Ca²⁺ nor byEDTA.

Amino acid analysis on the N-terminal amino acid sequence of the enzymeby “PROTEIN SEQUENCER MODEL 473A”, an apparatus of Applied Biosystems,Inc., Foster City, USA, revealed that the enzyme had a partial aminoacid sequence of SEQ ID NO:20, i.e., asparticacid-threonine-leucine-serine-glycine-valine-phenylalanine-histidine-glycine-prolineat the N-terminal region.

EXPERIMENT 20 Action on Saccharides

It was tested whether any saccharides can be used as substrates forα-isomaltosylglucosaccharide-forming enzyme. For the purpose, a solutionof maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose,maltoheptaose, isomaltose, isomaltotriose, panose, isopanose,α,α-trehalose, kojibiose, nigerose, neotrehalose, cellobiose,gentibiose, maltitol, maltotriitol, lactose, sucrose, erlose,selaginose, maltosyl glucoside, or isomaltosyl glucoside was prepared.

To each of the above solutions was added two units/g substrate of apurified specimen of α-isomaltosylglucosaccharide-forming enzyme fromeither Bacillus globisporus C9 strain obtained by the method inExperiment 4-2, Bacillus globisporus C11 strain obtained by the methodin Experiment 7-2, Bacillus globisporus N75 strain obtained by themethod in Experiment 11-2, or Arthrobacter globiformis A19 strainobtained by the method in Experiment 15-2, and the resulting eachsolution was adjusted to give a substrate concentration of 2% (w/v) andincubated at 30° C. and pH 6.0 for 24 hours, except for using pH 8.4 forthe enzyme from Arthrobacter globiformis A19 strain. The enzymesolutions before and after the enzymatic reactions were respectivelyanalyzed on TLC disclosed in Experiment 1 to confirm whether the enzymesacted on these substrates. The results are in Table 22.

TABLE 22 Enzymatic action Enzyme of Enzyme of Enzyme of Enzyme ofSubstrate C9 strain C11 strain N75 strain A19 strain Maltose + + + +Maltotriose ++ ++ ++ ++ Maltotetraose +++ +++ +++ +++ Maltopentaose ++++++ +++ +++ Maltohexaose +++ +++ +++ +++ Maltoheptaose +++ +++ +++ +++Isomaltose − − − − Isomaltotriose − − − − Panose − − − − Isopanose ++ ++++ ++ Trehalose − − − − Kojibiose + + + + Nigerose + + + +Neotrehalose + + + + Cellobiose − − − − Gentibiose − − − − Maltitol − −− − Maltotriitol + + + + Lactose − − − − Sucrose − − − − Erlose + + + +Selaginose − − − − Maltosyl glucoside ++ ++ ++ ++ Isomaltosyl glucoside− − − − Note: Before and after the enzymatic reaction, the symbols “−”,“+”, “++”, and “+++”, mean that it showed no change, it showed a slightreduction of the color spot of the substrate and the formation of otherreaction product, it showed a high reduction of the color spot of thesubstrate and the formation of other reaction product, and it showed asubstantial disappearance of the substrate spot and the formation ofother reaction product, respectively.

As evident from the Table 22, it was revealed that theα-isomaltosylglucosaccharide-forming enzymes well acted on saccharideshaving a glucose polymerization degree of at least three and having amaltose structure at their non-reducing ends, among the saccharidestested. It was also found that the enzymes slightly acted onsaccharides, having a glucose polymerization degree of two, such asmaltose, kojibiose, nigerose, neotrehalose, maltotriitol, and erlose.

EXPERIMENT 21 Reaction product from Maltooligosaccharide EXPERIMENT 21-1Preparation of Reaction Product

To an aqueous solution containing one percent (w/v) of maltose,maltotriose, maltotetraose, or maltopentaose as a substrate was added apurified specimen of α-isomaltosylglucosaccharide-forming enzymeobtained by the method in Experiment 7-2 in an amount of two units/gsolid, d.s.b., for the aqueous solutions of maltose and maltotriose; 0.2unit/g solid, d.s.b., for the aqueous solution of maltotetraose; and 0.1unit/g solid, d.s.b., for the aqueous solution of maltopentaose,followed by incubation at 35° C. and pH 6.0 for eight hours. After a10-min incubation at 100° C., the enzymatic reaction was suspended. Theresulting reaction solutions were respectively measured for saccharidecomposition on HPLC using “YMC PACK ODS-AQ303”, a column commercializedby YMC Co., Ltd., Tokyo, Japan, at a column temperature of 40° C. and aflow rate of 0.5 ml/min of water, and using as a detector “RI-8012”, adifferential refractometer commercialized by Tosoh Corporation, Tokyo,Japan. The results are in Table 23.

TABLE 23 Substrate Saccharide as Mal- reaction product tose MaltotrioseMaltotetraose Maltopentaose Glucose 8.5 0.1 0.0 0.0 Maltose 78.0 17.90.3 0.0 Maltotriose 0.8 45.3 22.7 1.9 Maltotetraose 0.0 1.8 35.1 19.2Maltopentaose 0.0 0.0 3.5 34.4 Maltohexaose 0.0 0.0 0.0 4.6 Isomaltose0.5 0.0 0.0 0.0 Glucosylmaltose 8.2 1.2 0.0 0.0 Glucosyl- 2.4 31.5 6.80.0 maltotriose X 0.0 2.1 30.0 11.4 Y 0.0 0.0 1.4 26.8 Z 0.0 0.0 0.0 1.7Others 0.6 0.1 0.2 0.0 Note: In the table, glucosylmaltose meansα-isomaltosylglucose alias 6²-O-α-glucosylmaltose or panose;glucosylmaltotriose means α-isomaltosylglucose alias6³-O-α-glucosylmaltotriose; X means the α-isomaltosylmaltotriose inExperiment 11-2, alias 6⁴-O-α-glucomaltotetraose; Y means theα-isomaltosylmaltotetraose in Experiment 11-2, alias6⁵-O-α-glucosylmaltopentaose; and Z means an unidentified saccharide.

As evident from the results in Table 23, it was revealed that, after theenzymatic action, glucose and α-isomaltosylglucose alias6²-O-α-glucosylmaltose or panose were mainly formed maltose as asubstrate; and maltose and α-isomaltosylglucose alias6³-O-α-glucosylmaltotriose were mainly formed along with small amountsof glucose, maltotetraose, α-isomaltosylglucose alias6²-O-α-glucosylmaltose or panose, and a product X. Also, it was revealedthat maltotriose and the product X were mainly formed from maltotetraoseas a substrate along with small amounts of maltose, maltopentaose,α-isomaltosylglucose alias 6³-O-α-glucosylmaltotriose; and a product Y;and that maltotetraose and the product Y were mainly formed frommaltopentaose as a substrate along with small amounts of maltotriose,maltohexaose, and the products X and Z. The product X as a main productfrom maltotetraose as a substrate and the product Y as a main productfrom maltopentaose as a substrate were respectively isolated andpurified as follows: The products X and Y were respectively purified onHPLC using “YMC PACK ODS-A R355-15S-15 12A”, a separatory HPLC columncommercialized by YMC Co., Ltd., Tokyo, Japan, to isolate the product Xhaving a purity of at least 99.9% from the reaction product frommaltotetraose in a yield of about 8.3%, d.s.b., and the product Y havinga purity of at least 99.9% from the reaction product from maltopentaosein a yield of about 11.5%, d.s.b.

EXPERIMENT 21-2 Structural Analysis on Reaction Product

The products X and Y, obtained by the method in Experiment 21-1, weresubjected to methyl analysis and NMR analysis in a usual manner. Theresults on their methyl analyses are in Table 24. Regarding the resultson their NMR analyses, FIG. 42 is a ¹H-NMR spectrum for the product Xand FIG. 43 is for the product Y. The ¹³C-NMR spectra for the products Xand Y are respectively FIGS. 44 and 45. The assignment of the products Xand Y are tabulated in Table 25.

TABLE 24 Analyzed Ratio methyl compound Product X Product Y2,3,4-Trimethyl compound 1.00 1.00 2,3,6-Trimethyl compound 3.05 3.982,3,4,6-Tetramethyl compound 0.82 0.85

TABLE 25 Glucose Carbon Chemical shift on NMR (ppm) number numberProduct X Product Y a 1a 100.8 100.8 2a 74.2 74.2 3a 75.8 75.7 4a 72.272.2 5a 74.5 74.5 6a 63.2 63.1 b 1b 102.6 102.6 2b 74.2 74.2 3b 75.875.7 4b 72.1 72.1 5b 74.0 74.0 6b 68.6 68.6 c 1c 102.3 102.3 2c 74.274.2 3c 76.0 76.0 4c 79.6 79.5 5c 73.9 73.9 6c 63.2 63.1 d 1d 102.2102.3 2d 74.0 (α), 74.4 (β) 74.2 3d 76.0 76.0 4d 79.8 79.5 5d 73.9 73.96d 63.2 63.1 e 1e 94.6 (α), 98.5 (β) 102.1 2e 74.2 (α), 76.7 (β) 74.0(α), 74.4 (β) 3e 75.9 (α), 78.9 (β) 76.0 4e 79.6 (α), 79.4 (β) 79.8 5e72.6 (α), 77.2 (β) 73.9 6e 63.4 (α), 63.4 (β) 63.1 f 1f 94.6 (α), 98.5(β) 2f 74.2 (α), 76.7 (β) 3f 76.0 (α), 78.9 (β) 4f 79.6 (α), 79.5 (β) 5f72.6 (α), 77.2 (β) 6f 63.3 (α), 63.3 (β)

Based on these results, the product X, formed from maltotetraose via theaction of the α-isomaltosylglucosaccharide-forming enzyme, was revealedas a pentasaccharide, in which a glucose residue is linked via theα-linkage to OH-6 of the glucose positioning at the non-reducing end ofmaltotetraose, i.e., α-isomaltosylmaltotriose alias6⁶-O-α-glucosylmaltotetraose, represented by Formula 1.α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-D-Glcp  Formula1:

The product Y formed from maltopentaose was revealed as ahexasaccharide, in which a glucosyl residue is linked via the α-linkageto OH-6 of the glucose at the non-reducing end of maltopentaose, i.e.,α-isomaltosylmaltotetraose alias 6⁵-O-α-glucosylmaltopentaose,represented by Formula 2.α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-D-Glcp  Formula2:

Based on these results, it was concluded thatα-isomaltosylglucosaccharide-forming enzyme acts onmaltooligosaccharides as indicated below:

-   -   (1) The enzyme acts on as a substrate maltooligosaccharides        having a glucose polymerization degree of at least two linked        via the α-1,4 linkage, and catalyzes the intermolecular        6-glucosyl-transferring reaction in such a manner of        transferring a glucosyl residue at the non-reducing end of a        maltooligosaccharide molecule to C-6 of the non-reducing end of        other maltooligosaccharide molecule to form both an        α-isomaltosylglucosaccharide alias        6-O-α-glucosylmaltooligosaccharide, having a 6-O-α-glucosyl        residue and an increased glucose polymerization degree by one as        compared with the intact substrate, and a maltooligosaccharide        with a reduced glucose polymerization degree by one as compared        with the intact substrate molecule; and    -   (2) The enzyme slightly catalyzes the 4-glucosyl-transferring        reaction and forms both a maltooligosaccharide molecule, having        an increased glucose polymerization degree by one as compared        with the intact substrate, and a maltooligosaccharide having a        reduced glucose polymerization degree by one as compared with        the intact substrate molecule.

EXPERIMENT 22 Test on Reducing-power Formation

The following test was carried out to study whetherα-isomaltosylglucosaccharide-formation enzyme had the ability of forminga reducing power. To a 1% (w/v) aqueous solution of maltotetraose as asubstrate was added 0.25 unit/g substrate, d.s.b., of either of purifiedspecimens of α-isomaltosylglucosaccharide-forming enzyme from Bacillusglobisporus C9 strain obtained by the method in Experiment 4-2, Bacillusglobisporus C11 strain obtained by the method in Experiment 7-2,Bacillus globisporus N75 strain obtained by the method in Experiment11-2, or Arthrobacter globiformis A19 strain obtained by the method inExperiment 15-2, and incubated at 35° C. and pH 6.0, except that pH 8.4was used for the enzyme from Arthrobacter globiformis A19 strain. Duringthe enzymatic reaction, a portion of each reaction solution was sampledat prescribed time intervals and measured for reducing powder afterkeeping the sampled solutions at 100° C. for 10 min to suspend theenzymatic reaction. Before and after the enzymatic reaction, thereducing saccharide content and the total sugar content wererespectively quantified by the Somogyi-Nelson's method and theanthrone-sulfuric acid reaction method. The percentage of formingreducing power was calculated by the following equation:

Equation:

$\begin{matrix}{{Percentage}\mspace{14mu}{of}\mspace{14mu}{forming}} \\{{reducing}\mspace{14mu}{power}\mspace{14mu}(\%)}\end{matrix} = {\left( {\frac{AR}{AT} - \frac{BR}{BT}} \right) \times 100}$

-   AR: Reducing sugar content after enzymatic reaction.-   AT: Total sugar content after enzymatic reaction.-   BR: Reducing sugar content before enzymatic reaction.-   BT: Total sugar content before enzymatic reaction.

The results are in Table 26.

TABLE 26 Percentage of forming Reaction reducing power (%) time Enzymeof Enzyme of Enzyme of Enzyme of (hour) C9 strain C11 strain N75 strainA19 strain 0 0.0 0.0 0.0 0.0 1 0.0 0.1 0.1 0.0 2 0.1 0.0 0.0 0.1 4 0.10.1 0.0 0.0 8 0.0 0.0 0.1 0.1

As evident from the results in Table 26, it was revealed thatα-isomaltosylglucosaccharide-forming enzyme did not substantiallyincrease the reducing power of the reaction product when acted onmaltotetraose as a substrate; the enzyme did not have any hydrolyzingactivity or had only an undetectable level of such activity.

EXPERIMENT 23 Test on Dextran Formation

To examine whether α-isomaltosylglucosaccharide-formation enzyme has theability of forming dextran, it was tested in accordance with the methodin Bioscience Biotechnology and Biochemistry, Vol. 56, pp. 169-173(1992). To a 1% (w/v) aqueous solution of maltotetraose as a substratewas added 0.25 unit/g substrate, d.s.b., of either of purified specimensof α-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporusC9 strain obtained by the method in Experiment 4-2, Bacillus globisporusC11 strain obtained by the method in Experiment 7-2, Bacillusglobisporus N75 strain obtained by the method in Experiment 11-2, orArthrobacter globiformis A19 strain obtained by the method in Experiment15-2, and incubated at 35° C. and pH 6.0, except that pH 8.4 was usedfor the enzyme from Arthrobacter globiformis A19 strain, for four oreight hours. After completion of the enzymatic reaction, the reactionwas suspended by heating at 100° C. for 15 min. Fifty microliters ofeach of the reaction mixtures were placed in a centrifugation tube andthen admixed and sufficiently stirred with 3-fold volumes of ethanol,followed by standing at 4° C. for 30 min. Thereafter, each mixturesolution was centrifuged at 15,000 rpm for five minutes and, afterremoving supernatant, the resulting sediment was admixed with onemilliliter of 75% ethanol solution and stirred for washing. Theresulting each solution was centrifuged to remove supernatant, dried invacuo, and then admixed and sufficiently stirred with one milliliter ofdeionized water. The total sugar content, in terms of glucose, of eachof the resulting solutions was quantified by the phenol-sulfuric acidmethod. As a control, the total sugar content was determined similarlyas in the above except for using either of purified specimens ofα-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporus C9strain, Bacillus globisporus C11 strain, Bacillus globisporus N75strain, and Arthrobacter globiformis A19 strain, which had beeninactivated at 100° C. for 10 min. The content of dextran formed wascalculated by the following equation.Content of dextran formed (mg/ml)=[(Total sugar content for testsample)]−[(Total sugar content for control sample)]×20  Equation

The results are in Table 27.

TABLE 27 Reaction Content of dextran formed (mg/ml) time Enzyme ofEnzyme of Enzyme of Enzyme of (hour) C9 strain C11 strain N75 strain A19strain 4 0.0 0.0 0.0 0.0 8 0.0 0.0 0.0 0.0

As evident from the results in Table 27, it was revealed thatα-isomaltosylglucosaccharide-forming enzyme did not substantially havethe action of forming dextran or had only an undetectable level of suchactivity because it did not form dextran when acted on maltotetraose.

EXPERIMENT 24 Transfer-acceptor Specificity

Using various saccharides, it was tested whether the saccharides wereused as transferring-acceptors for α-isomaltosylglucosaccharide-formingenzyme. A solution of D-glucose, D-xylose, L-xylose, D-galactose,D-fructose, D-mannose, D-arabinose, D-fucose, D-psicose, L-sorbose,L-rhamnose, methyl-α-glucopyranoside, methyl-β-glucopyranoside,N-acetyl-glucosamine, sorbitol, α,α-trehalose, isomaltose,isomaltotriose, cellobiose, gentibiose, glycerol, maltitol, lactose,sucrose, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or L-ascorbicacid was prepared. To each solution with a saccharide concentration of1.6% was added “PINE-DEX #100”, a partial starch hydrolyzate, as asaccharide donor, to give a concentration of 4%, and admixed with oneunit/g saccharide donor, d.s.b., of either of purified specimens ofα-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporus C9strain obtained by the method in Experiment 4-2, Bacillus globisporusC11 strain obtained by the method in Experiment 7-2, Bacillusglobisporus N75 strain obtained by the method in Experiment 11-2, orArthrobacter globiformis A19 strain obtained by the method in Experiment15-2, and incubated at 30° C. and pH 6.0 for 24 hours, except that pH8.4 was used for the enzyme from Arthrobacter globiformis A19 strain.The reaction mixtures of the post-enzymatic reactions were analyzed ongas chromatography (abbreviated as “GLC” hereinafter) formonosaccharides and disaccharides as acceptors, and on HPLC fortrisaccharides as acceptors to confirm whether these saccharides couldbe used as the transfer acceptors of the above enzymes. In the case ofperforming GLC, the following apparatuses and conditions were used: GLCapparatus, “GC-16A” commercialized by Shimadzu Corporation, Tokyo,Japan; column, a stainless-steel column, 3 mm in diameter and 2 m inlength, packed with 2% “SILICONE OV-17/CHROMOSOLV W”, commercialized byGL Sciences Inc., Tokyo, Japan; carrier gas, nitrogen gas at a flow rateof 40 ml/min under temperature conditions of increasing from 160° C. to320° C. at an increasing temperature rate of 7.5° C./min; and detection,a hydrogen flame ionization detector. In the case of performing HPLCanalysis, the following apparatuses and conditions were used: HPLCapparatus, “CCPD” commercialized by Tosoh Corporation, Tokyo, Japan;column, “ODS-AQ-303” commercialized by YMC Co., Ltd., Tokyo, Japan;eluent, water at a flow rate of 0.5 ml/min; and detection, adifferential refractometer. The results are in Table 28.

TABLE 28 Product of transferring reaction Enzyme of Enzyme of Enzyme ofEnzyme of Saccharide C9 strain C11 strain N75 strain A19 strainD-Glucose + + + + D-Xylose ++ ++ ++ + L-Xylose ++ ++ ++ +D-Galactose + + + ± D-Fructose + + + + D-Mannose − − − ± D-Arabinose ± ±± ± D-Fucose + + + ± D-Psicose + + + + L-Sorbose + + + + L-Rhamnose − −− − Methyl-α- ++ ++ ++ ++ glucopyranoside Methyl-β- ++ ++ ++ ++glucopyranoside N-Acetylglucosamine + + + − Sorbitol − − − − Trehalose++ ++ ++ ++ Isomaltose ++ ++ ++ + Isomaltotriose ++ ++ ++ ± Cellobiose++ ++ ++ ++ Gentibiose ++ ++ ++ + Glycerol + + + + Maltitol ++ ++ ++ ++Lactose ++ ++ ++ ++ Sucrose ++ ++ ++ ++ α-Cyclodextrin − − − −β-Cyclodextrin − − − − γ-Cyclodextrin − − − − L-Ascorbic acid + + + +Note: In the table, the symbols “−”, “±”, “+”, and “++” mean that nosaccharide-transferred product was detected through transfer reaction toacceptor; a saccharide-transferred product was detected in an amountless than one percent through transfer reaction to acceptor; asaccharide-transferred product was detected in an amount over onepercent but less than 10% through transfer reaction to acceptor; and asaccharide-transferred product was detected in an amount over tenpercent through transfer reaction to acceptor.

As evident from the results in Table 28, it was revealed thatα-isomaltosylglucosaccharide-forming enzymes utilizes different types ofsaccharides as transfer acceptors; theα-isomaltosylglucosaccharide-forming enzymes from C9, C11 and N75strains advantageously transfer a saccharide(s), particularly, toD-/L-xylose, methyl-α-glucopyranoside, methyl-β-glucopyranoside,α,α-trehalose, isomaltose, isomaltotriose, cellobiose, gentibiose,maltitol, lactose, and sucrose; then transfer to D-glucose, D-fructose,D-fucose, D-psicose, L-sorbose, N-acetylglucosamine, glycerol, andL-ascorbic acid; and further to D-arabinose. Particularly, theα-isomaltosylglucosaccharide-forming enzyme from A19 strain welltransfers a saccharide(s), specifically, to methyl-α-glucopyranoside,methyl-β-glucopyranoside, α,α-trehalose, cellobiose, maltitol, lactose,and sucrose; secondary transfers to D-glucose, D-/L-xylose, D-fructose,D-psicose, L-sorbose, isomaltose, gentibiose, glycerol, and L-ascorbicacid; and thirdly to D-galactose, D-mannose, D-arabinose, D-fucose, andisomaltotriose.

The properties of α-isomaltosylglucosaccharide-transferring enzyme asdescribed above were compared with those of a previously reported enzymehaving 6-glucosyl-transferring action; a dextrin dextranase disclosed in“Bioscience Biotechnology and Biochemistry”, Vol. 56, pp. 169-173(1992); and a transglucosidase disclosed in “Nippon Nogeikagaku Kaishi”,Vol. 37, pp. 668-672 (1963). The results are in Table 29.

TABLE 29 α-Isomaltosyl-glucosaccharide- Dextrin forming enzyme of thepresent invention dextranase Transglucosidase Property C9 strain C11strain N75 strain A19 strain Control Control Hydrolysis NegativeNegative Negative Negative Negative Positive activity Optimum pH 6.0-6.56.0 6.0 8.4 4.0 to 4.2 3.5 Inhibition Positive Positive PositivePositive Negative Negative by EDTA

As evident from Table 29, α-isomaltosylglucosaccharide-forming enzymehad outstandingly novel physicochemical properties completely differentfrom those of conventionally known dextrin dextranase andtransglucosidase.

EXPERIMENT 25 Formation of Cyclotetrasaccharide

The test on the formation of cyclotetrasaccharide byα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme was conducted using saccharides. Forthe test, it was prepared a solution of maltose, maltotriose,maltotetraose, maltopentaose, amylose, soluble starch, “PINE-DEX #100”(a partial starch hydrolyzate commercialized by Matsutani Chemical Ind.,Tokyo, Japan), or glycogen from oyster commercialized by Wako PureChemical Industries Ltd., Tokyo, Japan.

To each of these solutions with a concentration of 0.5%, one unit/gsolid, d.s.b., of a purified specimen ofα-isomaltosylglucosaccharide-forming enzyme from C11 strain obtained bythe method in Experiment 7-2 and 10 units/g solid, d.s.b., of a purifiedspecimen of α-isomaltosyl-transferring enzyme from C11 strain obtainedby the method in Experiment 7-3, and the resulting mixture was subjectedto an enzymatic reaction at 30° C. and pH 6.0. The enzymatic conditionswere the following four systems:

-   -   (1) After the α-isomaltosylglucosaccharide-forming enzyme was        allowed to act on a saccharide solution for 24 hours, the enzyme        was inactivated by heating, and then the        α-isomaltosyl-transferring enzyme was allowed to act on the        resulting mixture for 24 hours and then inactivated by heating;    -   (2) After the α-isomaltosylglucosaccharide-forming enzyme and        the α-isomaltosyl-transferring enzyme were simultaneously        allowed to act on a saccharide solution for 24 hours, the        enzymes were inactivated by heating;    -   (3) After only the α-isomaltosylglucosaccharide-forming enzyme        was allowed to act on a saccharide solution for 24 hours, the        enzyme was inactivated by heating; and    -   (4) After only the α-isomaltosyl-transferring enzyme was allowed        to act on a saccharide solution for 24 hours, the enzyme was        inactivated by heating.

To determine the formation level of cyclotetrasaccharide in eachreaction mixture after the inactivation of enzyme(s) by heating, thereaction mixture was treated with α-glucosidase and glucoamylasesimilarly as in Experiment 1 to hydrolyze the remaining reducingoligosaccharides, followed by the quantitation of cyclotetrasaccharideon HPLC. The results are in Table 30.

TABLE 30 Yield of cyclotetrasaccharide (%) Substrate A B C D Maltose 4.04.2 0.0 0.0 Maltotriose 10.2 12.4 0.0 0.0 Maltotetraose 11.3 21.5 0.00.0 Maltopentaose 10.5 37.8 0.0 0.0 Amylose 3.5 31.6 0.0 0.0 Solublestarch 5.1 38.2 0.0 0.0 Partial starch 6.8 63.7 0.0 0.0 hydrolyzateGlycogen 10.2 86.9 0.0 0.0 Note: The symbols “A”, “B”, “C” and “D” meanthat α-isomaltosylglucosaccharide-forming enzyme was first allowed toact on a substrate and then α-isomaltosyl-transferring enzyme wasallowed acted on the substrate, the α-isomaltosylglucosaccharide-formingenzyme and α-isomaltosyl-transferring enzyme were allowed to coact on asubstrate, only α-isomaltosylglucosaccharide-forming enzyme was allowedto act on a substrate, and only α-isomaltosyl-transferring enzyme wasallowed to act on a substrate.

As evident from the results in Table 30, no cyclotetrasaccharide wasformed from any of the saccharides tested by the single action of eitherα-isomaltosylglucosaccharide-forming enzyme orα-isomaltosyl-transferring enzyme, but cyclotetrasaccharide was formedby the coaction of these enzymes. It was revealed that the formationlevel of cyclotetrasaccharide was relatively low, i.e., about 11% orlower, when α-isomaltosyl-transferring enzyme was allowed to act on thesaccharides after the action of α-isomaltosylglucosaccharide-formingenzyme, while the formation level was increased when the enzymes wereallowed to coact on any of the saccharides tested, particularly, it wasincreased to about 87% and about 64% when the enzymes were allowed tocoact on glycogen and partial starch hydrolyzate, respectively.

Based on the reaction properties of α-isomaltosylglucosaccharide-formingenzyme and α-isomaltosyl-transferring enzyme, the formation mechanism ofcyclotetrasaccharide by the coaction of these enzymes is estimated asfollows:

-   -   (1) α-Isomaltosylglucosaccharide-forming enzyme acts on a        glucose residue at the non-reducing end of an α-1,4 glucan chain        of glycogen and partial starch hydrolyzates, etc., and        intermolecularly transfers the glucose residue to OH-6 of the        glucose residue at the non-reducing end of other α-1,4 glucan        chain of glycogen and partial starch hydrolyzates, etc., to form        an α-1,4 glucan chain having an α-isomaltosyl residue at the        non-reducing end;    -   (2) α-Isomaltosyl-transferring enzyme acts on the α-1,4 glucan        chain having an α-isomaltosyl residue at the non-reducing end        and intermolecularly transfers the isomaltosyl residue to C-3 of        a glucose residue at the non-reducing end of other α-1,4 glucan        chain having an isomaltosyl residue at the non-reducing end to        form an α-1,4 glucan chain having an isomaltosyl-1,3-isomaltosyl        residue at the non-reducing end;    -   (3) Then, α-isomaltosyl-transferring enzyme acts on the α-1,4        glucan chain having an isomaltosyl-1,3-isomaltosyl residue at        the non-reducing end and releases the        isomaltosyl-1,3-isomaltosyl residue from the α-1,4 glucan chain        via the intramolecular transferring reaction to cyclize the        released isomaltosyl-1,3-isomaltosyl residue into        cyclotetrasaccharide;    -   (4) From the released α-1,4 glucan chain, cyclotetrasaccharide        is successively formed through the sequential steps (1) to (3).        Thus, it is estimated that the coaction of        α-isomaltosylglucosaccharide-forming enzyme and        α-isomaltosyl-transferring enzyme increases the formation of        cyclotetrasaccharide in such a cyclic manner as indicated above.

EXPERIMENT 26 Influence of Liquefaction Degree of Starch

A 15% corn starch suspension was prepared, admixed with 0.1% calciumcarbonate, adjusted to pH 6.0, and then mixed with 0.2 to 2.0% per gramstarch of “TERMAMYL 60L™”, an α-amylase specimen commercialized by NovoIndutri A/S, Copenhagen, Denmark, followed by an enzymatic reaction at95° C. for 10 min. Thereafter, the reaction mixture was autoclaved at120° C. for 20 min, promptly cooled to about 35° C. to obtain aliquefied starch solution with a DE (dextrose equivalent) of 3.2 to20.5. To the liquefied starch solution were added two units/g solid,d.s.b., of a purified specimen of α-isomaltosylglucosaccharide-formingenzyme from C11 strain obtained by the method in Experiment 7-2, and 20units/g solid, d.s.b., of a purified specimen ofα-isomaltosyl-transferring enzyme from C11 strain obtained by the methodin Experiment 7-3, followed by an incubation at 35° C. for 24 hours.After completion of the reaction, the reaction mixture was heated at100° C. for 15 min to inactivate the remaining enzymes. Then, thereaction mixture thus obtained was treated with α-glucosidase andglucoamylase similarly as in Experiment 1 to hydrolyze the remainingreducing oligosaccharides, followed by quantifying the formedcyclotetrasaccharide on HPLC. The results are in Tale 31.

TABLE 31 Amount of α-amylase Yield of per starch (%) DEcyclotetrasaccharide (%) 0.2 3.2 54.5 0.4 4.8 50.5 0.6 7.8 44.1 1.0 12.539.8 1.5 17.3 34.4 2.0 20.5 30.8

As evident from the results in Table 31, it was revealed that theformation of cyclotetrasaccharide by the coaction ofα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme is influenced by the liquefactiondegree of starch, i.e., the lower the liquefaction degree or the lowerthe DE, the more the yield of cyclotetrasaccharide from starchincreases. On the contrary, the higher the liquefaction degree or thehigher the DE, the lower the yield of cyclotetrasaccharide from starchdecreases. It was revealed that a suitable liquefaction degree is a DEof about 20 or lower, preferably, a DE of about 12 or lower, morepreferably, a DE of about five or lower.

EXPERIMENT 27 Influence of the Concentration of Partial StarchHydrolyzate

Aqueous solutions of “PINE-DEX #100”, a partial starch hydrolyzate witha DE of about two to about five, having a final concentration of 0.5 to40%, were prepared and respectively admixed with one unit/g solid,d.s.b., of the purified specimen of α-isomaltosylglucosaccharide-formingenzyme from C11 strain obtained by the method in Experiment 7-2 and 10units/g solid, d.s.b., of a purified specimen ofα-isomaltosyl-transferring enzyme from C11 strain obtained by the methodin Experiment 7-3, followed by the coaction of these enzymes at 30° C.and pH 6.0 for 48 hours. After completion of the enzymatic reaction, thereaction mixture was heated at 100° C. for 15 min to inactivate theremaining enzymes, and then treated with α-glucosidase and glucoamylasesimilarly as in Experiment 1 to hydrolyze the remaining reducingoligosaccharides, followed by quantifying the formedcyclotetrasaccharide on HPLC. The results are in Table 32.

TABLE 32 Concentration of Yield of PINE-DEX (%) cyclotetrasaccharide (%)0.5 63.6 2.5 62.0 5 60.4 10 57.3 15 54.6 20 51.3 30 45.9 40 35.9

As evident from the results in Table 32, the yield ofcyclotetrasaccharide was about 64% at a low concentration of 0.5%, whileit was about 40% at a high concentration of 40%. The fact indicates thatthe yield of cyclotetrasaccharide increases depending on theconcentration of partial starch hydrolyzate as a substrate. The resultrevealed that the yield of cyclotetrasaccharide increased as thedecrease of concentration of partial starch hydrolyzate.

EXPERIMENT 28 Influence of the Addition of CyclodextrinGlucanotransferase

A 15% aqueous solution of “PINE-DEX #100”, a partial starch hydrolyzate,was prepared and admixed with one unit/g solid, d.s.b., of the purifiedspecimen of α-isomaltosylglucosaccharide-forming enzyme from C11 strainobtained by the method in Experiment 7-2, 10 units/g solid, d.s.b., of apurified specimen of α-isomaltosyl-transferring enzyme from C11 strainobtained by the method in Experiment 7-3, and 0 to 0.5 unit/g solid,d.s.b., of CGTase from a microorganism of the species Bacillusstearothermophilus, followed by the coaction of these enzymes at 30° C.and pH 6.0 for 48 hours. After completion of the reaction, the reactionmixture was heated at 100° C. for 15 min to inactivate the remainingenzymes, and then treated with α-glucosidase and glucoamylase similarlyas in Experiment 1 to hydrolyze the remaining reducing oligosaccharides,followed by quantifying the formed cyclotetrasaccharide on HPLC. Theresults are in Table 33.

TABLE 33 Amount of CGTase added Yield of (unit) cyclotetrasaccharide (%)0 54.6 2.5 60.1 5 63.1 10 65.2

As evident from the Table 33, it was revealed that the addition ofCGTase increased the yield of cyclotetrasaccharide.

EXPERIMENT 29 Preparation of Isomaltose-releasing Enzyme

A liquid medium, consisting of 3.0% (w/v) of dextran, 0.7% (w/v) ofpeptone, 0.2% (w/v) of dipotassium phosphate, 0.05% (w/v) of magnesiumsulfate heptahydrate, and water, was placed in 500-ml Erlenmeyer flasksin a volume of 100 ml each, autoclaved at 121° C. for 20 minutes forsterilization, cooled, inoculated with a stock culture of Arthrobacterglobiformis T6 strain (IAM 12103), and incubated at 27° C. for 48 hoursunder rotary shaking conditions of 230 rpm to obtain a seed culture.About 20 L of a fresh preparation of the same nutrient culture medium asused in the above culture were placed in a 30-L fermentor, sterilized byheating, cooled to 27° C., inoculated with 1% (v/v) of the seed culture,and further incubated for about 72 hours while stirring underaeration-agitation conditions at 27° C. and pH 6.0 to 8.0. Aftercompletion of the culture, the resultant culture, having about 16.5units/ml of isomaltodextranase activity, was centrifuged at 10,000 rpmfor 30 min to obtain about 18 L of a supernatant, having about 16units/ml of the enzyme, in a total enzyme activity of about 288,000units. The activity of isomaltodextranase was assayed by providing, as asubstrate solution, four milliliters of 1.25% (w/v) of an aqueousdextran solution in the form of 0.1M acetate buffer (pH 5.5), adding onemilliliter of an enzyme solution, subjecting the mixture to an enzymaticreaction at 40° C. for 20 min, sampling one milliliter of the reactionmixture, adding two milliliters of the Somogyi copper solution tosuspend the enzymatic reaction, and quantifying the reducing power ofthe formed isomaltose by the Somogyi-Nelson's method. One unit activityof isomaltodextranase is defined as the enzyme amount that forms areducing power corresponding to one micromole of isomaltose per minute.About 18 L of the culture supernatant were concentrated with a UFmembrane into about two liters, salted out in an 80% ammonium sulfatesolution, and allowed to stand at 4° C. for 24 hours. The resultingprecipitate was collected by centrifugation at 10,000 rpm for 30 min,dissolved in 5 mM phosphate buffer (pH 6.8), and dialyzed against afresh preparation of the same buffer as used in the above to obtainabout 400 ml of a dialyzed solution. The solution as a crude enzymesolution thus obtained was fed to ion-exchange chromatography using twoliters of “SEPABEADS FP-DA13” gel. The component with isomaltodextranaseactivity did not adsorb on the gel and it was eluted in non-adsorbedfractions. The non-adsorbed fractions with the desired enzyme activitywere collected, pooled, salted out in an 80% ammonium sulfate solution,and allowed to stand at 4° C. for 24 hours. The resulting precipitatewas collected by centrifugation at 10,000 rpm for 30 min, dissolved in 5mM phosphate buffer (pH 6.8), and dialyzed against a fresh preparationof the same buffer as used in the above to obtain about 500 ml of adialyzed solution having an isomaltodextranase activity of 161,000units.

EXPERIMENT 30 Preparation of Isomaltose fromα-isomaltosylglucosaccharide and Cyclotetrasaccharide

To a 0.2% aqueous solution of panose, α-isomaltosylmaltose,α-isomaltosyltriose, α-isomaltosyltetraose, or cyclotetrasaccharide wasadded 100 units/g solid, d.s.b., of an isomaltodextranase specimen,obtained by the method in Experiment 29, where 3,000 units/g solid,d.s.b., of the specimen was also used for the aqueous solution withcyclotetrasaccharide. The mixture was subjected to an enzymatic reactionat 40° C. and pH 5.5 for 24 hours and heated at 100° C. for 20 min tosuspend the enzymatic reaction. The saccharide composition of theresulting mixture was analyzed on HPLC using column of “MCIGEL CK04SS”,a column commercialized by Mitsubishi Chemical Industries, Ltd., Tokyo,Japan; an inner column temperature of 80° C.; a flow rate of 0.5 ml/minof water as an eluate; and a detector of “RI-8012”, a differentialrefractometer commercialized by Tosoh Corporation, Tokyo, Japan. Theresults are in Table 34.

TABLE 34 Saccharide as reaction product Enzyme (peak area (%) on HPLC)Substrate (unit) G1 IM G2 G3 G4 A IMG1 100 35 65 0 0 0 0 IMG2 100 0 5149 0 0 0 IMG3 100 0 41 0 59 0 0 IMG4 100 0 35 0 0 65 0Cyclotetrasaccharide 100 0 22 0 0 0 78 3,000 0 100 0 0 0 0 Note: In thetable, the symbols “IMG1”, “IMG2”, “IMG3” and “IMG4” mean panose,α-isomaltosylmaltose, α-isomaltoglucotriose, and isomaltoglucotetraose,respectively. The symbols “G1”, “G2”, “G3” and “G4” mean glucose,isomaltose, maltose, maltotriose, and maltotetraose, respectively. Thesymbol “A” means an intermediate formed during the formation ofisomaltose from cyclotetrasaccharide.

As evident from the results in Table 34, it was revealed that, whenisomaltodextranase was allowed to act on α-isomaltosylglucosaccharides,only glucose and isomaltose were formed from panose as a substrate; onlyisomaltose and maltose were formed from α-isomaltosylmaltose as asubstrate; only isomaltose and maltotriose were formed fromα-isomaltosyltriose; and only isomaltose and maltotetraose were formedfrom α-isomaltosyltetraose as a substrate. It was also found that onlyisomaltose was formed via the product “A” from cyclotetrasaccharide as asubstrate.

Then, the purification and isolation of the above-identified product Awere conducted as follows: The product A was subjected to “YMC-PACKODS-AR355-15S-15 12A”, a separatory HPLC column commercialized by YMCCo., Ltd., Tokyo, Japan, for purifying and isolating. Thus, the productA with a purity of at least 98.2% was obtained in a yield of about 7.2%from the reaction product of cyclotetrasaccharide.

The product A was subjected to methyl analysis and NMR analysis in ausual manner. The result on the methyl analysis is in Table 35. Whilethe results on the NMR analyses are respectively in FIG. 46 for ¹H-NMRspectrum and in FIG. 47 for ¹³C-NMR spectrum. The data on assignment ofthe product A is tabulated in Table 36.

TABLE 35 Analyzed methyl compound Composition ratio 2,3,4-Trimethylcompound 2.00 2,3,6-Trimethyl compound 0.92 2,3,4,6-Tetramethyl compound0.88

TABLE 36 Glucose No. Carbon No. NMR chemical shift (ppm) a 1a 100.7 2a74.2 3a 75.8 4a 72.3 5a 74.5 6a 63.2 b 1b 102.1 2b 74.3 3b 75.9 4b 72.65b 74.2 6b 68.0 c 1c 100.6 2c 72.8 3c 83.0 4c 72.0 5c 73.1 6c 62.9 e 1e94.9(α), 98.8(β) 2e 74.1(α), 76.6(β) 3e 75.8(α), 78.7(β) 4e 72.1(α),72.1(β) 5e 72.6(α), 76.9(β) 6e 68.3(α), 68.3(β)

From these results, the product A, formed as an intermediate during theformation of isomaltose from cyclotetrasaccharide by the action ofisomaltodextranase, was revealed as a tetrasaccharide in the form of aring-opened cyclotetrasaccharide, formed as a result of the hydrolysisof any one of the 1,3-linkages of cyclotetrasaccharide, represented byFormula 3, i.e.,α-glucosyl-(1→6)-α-glucosyl-(1→3)-α-glucosyl-(1→6)-glucose (orring-opened tetrasaccharide).α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp  Formula 3:

Based on these results, it can be concluded that the mechanism of theaction of isomaltodextranase on α-isomaltosylglucosaccharide is asfollows:

Isomaltodextranase acts on an α-isomaltosylglucosaccharide, having a6-O-α-glucosyl group at the non-reducing end, as a substrate, andspecifically hydrolyzes the α-1,4 linkage between the isomaltosylresidue at the non-reducing end and the resting glucose ormaltooligosaccharide residue to form isomaltose and glucose or amaltooligosaccharide. Then the enzyme also acts on cyclotetrasaccharideas a substrate and hydrolyzes the α-1,3 linkage for ring-opening to formring-opened cyclotetrasaccharide as an intermediate, and further acts onthe formed ring-opened cyclotetrasaccharide and hydrolyzes the α-1,3linkage thereof to form isomaltose.

EXPERIMENT 31 Formation of Isomaltose from Different Substrates

Using different saccharides, the formation mechanism of the action ofα-isomaltosylglucosaccharide-forming enzyme and isomaltodextranase wasexamined. Maltose, maltotriose, maltotetraose, maltopentaose,maltohexaose, maltoheptaose, amylose, or “PINE-DEX #100”, a partialstarch hydrolyzate commercialized by Matsutani Chemical Ind., Tokyo,Japan, was dissolved in water to give a final concentration of fivepercent. Also, calcium chloride was dissolved in water to give a finalconcentration of 1 mM. To each of the above aqueous solutions 0.2 unit/gsolid, d.s.b., of the purified specimen ofα-isomaltosylglucosaccharide-forming enzyme from C11 strain obtained inExperiment 7-2, and 100 units/g solid, d.s.b., of an isomaltodextranasespecimen obtained by the method in Experiment 29, followed by anenzymatic reaction at 40° C. and pH 5.5. The reaction conditions usedwere the following two systems:

(1) After contacting the α-isomaltosylglucosaccharide-forming enzymewith any of the substrates for 65 hours, the enzyme was inactivated byheating, then the isomaltodextranase was allowed to act on the resultingmixture for 65 hours and inactivated by heating.

(2) After contacting the α-isomaltosylglucosaccharide-forming enzyme andthe isomaltodextranase with any of the substrates in combination for 65hours, the enzymes were inactivated by heating.

The resulting heated reaction mixtures were assayed for isomaltose yieldon HPLC. The results are in Table 37:

TABLE 37 Yield of isomaltose (%) Substrate Sequential use* Combinationuse** Maltose 6.6 7.0 Maltotriose 15.7 18.7 Maltotetraose 15.8 45.4Maltopentaose 15.3 55.0 Maltohexaose 10.1 58.1 Maltoheptaose 8.5 63.6Amylose 4.0 64.9 Partial starch hydrolyzate 3.8 62.7 Note: The symbols“*” and “**” mean that α-isomaltosylglucosaccharide-forming enzyme andisomaltodextranase were allowed to act on a substrate in this order andin combination, respectively.

As evident from the results in Table 37, all of the saccharides testedformed isomaltose through the action ofα-isomaltosylglucosaccharide-forming enzyme and isomaltodextranase. Itwas revealed that the sequential use ofα-isomaltosylglucosaccharide-forming enzyme and isomaltodextranase inthis order only gave a low yield of isomaltose as low as less than about15%, while the combination use of the enzymes gave an improved yield ofisomaltose, particularly, up to a high yield of 60% or higher ofisomaltose when the enzymes were allowed to coact on maltoheptaose,amylose, or partial starch hydrolyzate. The isomaltose formationmechanism by the combination use of α-isomaltosylglucosaccharide-formingenzyme and isomaltodextranase is speculated as follows based on theirenzymatic reaction properties:

(1) α-Isomaltosylglucosaccharide-forming enzyme acts on the glucoseresidue at the non-reducing end of an α-1,4 glucan chain such as ofamylose and partial starch hydrolyzates, and intermolecularly transfersthe glucose residue to the hydroxyl group at C-6 of the glucose residueat the non-reducing end of another α-1,4 glucan chain to form an α-1,4glucan chain having an α-isomaltosyl group at the non-reducing end.

(2) Isomaltodextranase acts on the formed α-1,4 glucan chain, having anα-isomaltosyl group at the non-reducing end, and hydrolyzes the α-1,4linkage between the isomaltosyl group and the resting α-1,4 glucan chainto form/release isomaltose and an α-1,4 glucan chain with a reducedglucose polymerization degree by two.

(3) The released α-1,4 glucan chain sequentially receives the enzymaticreactions of (1) and (2) and forms another isomaltose.

As explained above, it can be speculated that, when used in combination,α-Isomaltosylglucosaccharide-forming enzyme and isomaltodextranaserepeatedly act on their substrates to form isomaltose and increase theyield.

EXPERIMENT 32 Effect of the Addition of Isoamylase

An aqueous solution of “PINE-DEX #100”, a partial starch hydrolyzate,with a final concentration of five percent and 1 mM calcium chloride,was prepared, admixed with 0.2 unit/g starch, d.s.b., of the purifiedspecimen of α-isomaltosylglucosaccharide-forming enzyme from C11 strainobtained in Experiment 7-2, 100 units/g starch, d.s.b., of anisomaltodextranase specimen obtained by the method in Experiment 29, and0 to 250 units/g starch, d.s.b., of an isoamylase specimen of amicroorganism of the species Pseudomonas amyloderamosa commercialized byHayashibara Biochemical Laboratories, Inc., Okayama, Japan, followed byan enzymatic reaction at 40° C. and pH 5.5 for 65 hours. Thereafter, theresulting mixture was heated at 100° C. for 15 min to inactivate theremaining enzymes. The formed isomaltose was quantified by HPLC. Theresults are in Table 38.

TABLE 38 Isoamylase added Yield of isomaltose (unit) (%) 0 62.7 50 65.1250 71.1

As evident from the results in Table 38, it was revealed that theaddition of isoamylase increases the yield of isomaltose.

EXPERIMENT 33 Influence of the Concentration of Partial StarchHydrolyzate

Eight types of aqueous solutions, having different concentrations of“PINE-DEX #100”, a partial starch hydrolyzate, with a DE of about two toabout five, having a final concentration of 1 to 40%, and containing 1mM calcium chloride, were prepared, admixed with 0.2 unit/g starch,d.s.b., of the purified specimen of α-isomaltosylglucosaccharide-formingenzyme from C11 strain obtained in Experiment 7-2, 100 units/g starch,d.s.b., of an isomaltodextranase specimen obtained by the method inExperiment 29, and 250 units/g starch, d.s.b., of an isoamylase specimenof a microorganism of the species Pseudomonas amyloderamosacommercialized by Hayashibara Biochemical Laboratories, Inc., Okayama,Japan, followed by an enzymatic reaction at 40° C. and pH 5.5 for 65hours. Thereafter, the resulting mixture was heated at 100° C. for 15min to inactivate the remaining enzymes. The formed isomaltose wasquantified by HPLC. The results are in Table 39.

TABLE 39 Concentration of “PINE DEX 100” Yield of isomaltose (%) (%) 173.0 2.5 72.8 5 71.1 10 67.0 15 63.7 20 60.7 30 55.4 40 50.7

As evident from the results in Table 39, it was revealed that the yieldof isomaltose increased up to about 73% at a low concentration of onepercent of partial starch hydrolyzate, but decreased to about 51% at aconcentration of 40% of partial starch hydrolyzate, meaning that theyield of isomaltose varies depending on the concentration of partialstarch hydrolyzate as a substrate.

EXPERIMENT 34 Influence of the Degree of Liquefied Starch

A 15% corn starch suspension was prepared, admixed with 0.1% calciumcarbonate, adjusted to pH 6.0, and then mixed with 0.2 to 2.0% per gramstarch of “TERMAMYL 60L™”, an α-amylase specimen commercialized by NovoIndutri A/S, Copenhagen, Denmark, followed by an enzymatic reaction at95° C. for 10 min. Thereafter, the reaction mixture was autoclaved at120° C., promptly cooled to about 40° C. to obtain a liquefied starchsolution with a DE of 3.2 to 20.5. The liquefied starch solution wasadjusted to give a final starch concentration of 5% and to pH 5.5, andthen mixed with 0.2 unit/g solid, d.s.b., of a purified specimen ofα-isomaltosylglucosaccharide-forming enzyme from C11 strain obtained bythe method in Experiment 7-2, 100 units/g solid, d.s.b., of a purifiedspecimen of isomaltodextranase obtained by the method in Experiment 29,and 250 units/g solid, d.s.b., of an isoamylase specimen fromPseudomonas amyloderamosa commercialized by Hayashibara Biochemicallaboratories, Inc., Okayama, Japan, followed by an incubation at 40° C.for 65 hours. After completion of the reaction, the reaction mixture washeated at 100° C. for 15 min to inactivate the remaining enzymes. Theformed isomaltose was quantified by HPLC. The results are in Table 40.

TABLE 40 Amount of α-amylase Yield of per g starch (%) DE isomaltose (%)0.2 3.2 71.5 0.4 4.8 71.0 0.6 7.8 66.2 1.0 12.5 59.8 1.5 17.3 53.2 2.020.5 47.9

As evident from the results in Table 40, it was revealed that theformation of isomaltose by the coaction ofα-isomaltosylglucosaccharide-forming enzyme and isomaltodextranase isinfluenced by the liquefaction degree of starch, i.e., the lower theliquefaction degree or the lower the DE, the higher the yield ofisomaltose from starch increases. On the contrary, the higher theliquefaction degree or the higher the DE, the lower the yield ofisomaltose from starch decreases. It was revealed that a suitableliquefaction degree is a DE of about 20 or lower, preferably, a DE ofabout 12 or lower, more preferably, a DE of about five or lower.

EXPERIMENT 35 Effect of the Addition of CGTase and Glucoamylase

An aqueous solution, containing 20% of “PINE-DEX #100”, a partial starchhydrolyzate, and 1 mM calcium chloride, was prepared, mixed with 0.2unit/g solid, d.s.b., of a purified specimen ofα-isomaltosylglucosaccharide-forming enzyme from C11 strain obtained bythe method in Experiment 7-2, 100 units/g solid, d.s.b., of a purifiedspecimen of isomaltodextranase obtained by the method in Experiment 29,and 0 to 0.5 unit/g solid, d.s.b., of a CGTase specimen from Bacillusstearothermophilus commercialized by Hayashibara Biochemicallaboratories, Inc., Okayama, Japan, followed by incubating the mixtureat 40° C. and pH 5.5 for 65 hours and heating the resulting mixture at100° C. for 15 min to inactivate the remaining enzymes. To the mixturethus obtained was added 20 units/g starch, d.s.b., of “XL-4™”, aglucoamylase specimen commercialized by Nagase Biochemicals, Ltd.,Kyoto, Japan, incubated at 50° C. for 24 hours, and heated at 100° C.for 20 min to inactivate the remaining enzyme. The formed isomaltose wasquantified on HPLC. The results are in Table 41.

TABLE 41 Amount of CGTase added (unit/g solid, d.s.b.) Yield ofisomaltose (%) 0 60.7 0.1 62.9 0.25 65.0 0.5 66.4

As evident from the results in Table 41, it was revealed that theaddition of CGTase to the enzymatic reaction system ofisomaltodextranase and α-isomaltosylglucosaccharide-forming enzymeincreased the yield of isomaltose. In the above enzymatic reactionsystem, the glucoamylase was used to form isomaltose from saccharides,composed of isomaltose linked with one or more D-glucose residues, andto release the D-glucose residue(s) therefrom, resulting in an increasedyield of isomaltose.

EXPERIMENT 36 Formation of Isomaltose

About one hundred liters of an aqueous solution of phytoglycogen fromcorn, commercialized by Q.P. Corporation, Tokyo, Japan, were adjusted togive a concentration of 4% (w/v) and pH 6.0, heated to 30° C., admixedwith one unit/g solid, d.s.b., of a purified specimen ofα-isomaltosylglucosaccharide-forming enzyme from C11 strain obtained bythe method in Experiment 7-2, 10 units/g solid, d.s.b., of a purifiedspecimen of α-isomaltosyl-transferring enzyme from C11 strain obtainedby the method in Experiment 7-3, followed by incubating the mixture for48 hours and heating the resulting mixture at 100° C. for 10 min toinactivate the remaining enzymes. The mixture thus obtained was sampledfor quantifying the yield of cyclotetrasaccharide on HPLC, revealingthat it had about 84% of cyclotetrasaccharide in terms of sugarcomposition, where HPLC was carried out using “SHOWDEX KS-801™ column”,Showa Denko K.K., Tokyo, Japan, at a column temperature of 60° C. and aflow rate of 0.5 ml/min of water, and using “RI-8012™”, a differentialrefractometer commercialized by Tosoh Corporation, Tokyo, Japan. Theabove mixture was adjusted to pH 5.0 and 45° C., admixed with 1,500units/g starch, d.s.b. of “TRANSGLUCOSIDASE L AMANO™”, an α-glucosidasecommercialized by Amano Pharmaceutical Co., Ltd., Aichi, Japan, and 75units/g starch, d.s.b., of “XL-4™”, a glucoamylase specimencommercialized by Nagase Biochemicals, Ltd., Kyoto, Japan, incubated for24 hours to hydrolyze the remaining reducing oligosaccharides, etc. Theresulting mixture was adjusted to pH 5.8, kept at 90° C. for one hour toinactivate the remaining enzymes, and filtered to remove insolublesubstances. The filtrate was concentrated to give a concentration ofabout 16% with “HOLLOSEP® HR 5155PI”, a reverse osmotic membrane, ToyoboCo., Ltd., Tokyo, Japan, and in a usual manner decolored, desalted,filtered, and concentrated to obtain about 6.2 kg of a saccharidesolution with a solid content of about 3,700 g, d.s.b. The saccharidesolution was fed to a column packed with about 225 L of “AMBERLITECR-1310 (Na⁺-form)”, a strong-acid cation exchange resin commercializedby Japan Organo Co., Ltd., Tokyo, Japan, and chromatographed at a columntemperature of 60° C. and a flow rate of about 45 L/h. While thesaccharide composition of eluate from the column was monitoring by theabove-identified HPLC, fractions of cyclotetrasaccharide with a purityof at least 98% were collected, and in a usual manner desalted,decolored, filtered, and concentrated to obtain about 7.5 kg of asaccharide solution with a solid content of about 2,500 g, d.s.b. HPLCanalysis for saccharide composition of the solution thus obtainedrevealed that it contained cyclotetrasaccharide with a purity of about99.5%. The resulting saccharide solution with cyclotetrasaccharide wasconcentrated into an about 50% solution by an evaporator, and about fivekilograms of which were placed in a cylindrical plastic vessel, cooledfrom 65° C. to 20° C. over about 20 hours under gentle stirringconditions to crystallize cyclotetrasaccharide. Then, the resultingmassecuite was centrifugally separated to collect 1,360 g of crystallinecyclotetrasaccharide by wet weight, and dried at 60° C. for three hoursto obtain 1,170 g of a crystalline cyclotetrasaccharide powder. HPLCanalysis for saccharide composition of the powder revealed that it had apurity of cyclotetrasaccharide crystal as high as at least about 99.9%.

The above crystalline cyclotetrasaccharide powder was dissolved indeionized water, adjusted to give a concentration of one percent, pH 5.5and 50° C., admixed with 500 units/g solids, d.s.b., of anisomaltodextranase specimen prepared by the method in Experiment 29, andenzymatically reacted at pH 5.5 and 50° C. for 70 hours. Thereafter, theresulting mixture was heated to and kept at 95° C. for 10 min, cooled,and filtered. The filtrate was in a usual manner decolored with anactivated charcoal, desalted for purification with ion-exchange resinsin H- and OH-forms, and concentrated to give a concentration of about50%. Thus, a high isomaltose content syrup was obtained in a yield ofabout 95%, d.s.b., to the solid contents. HPLC analysis for saccharidecomposition of the syrup revealed that it contained 96.1% of isomaltose,2.8% of ring-opened tetrasaccharide, and 1.1% of other saccharides.

Four hundred grams of the above syrup were in a usual manner placed inan autoclave with 0.1 g/solids, d.s.b., of “N154™”, analkaline-developed Raney nickel catalyst commercialized by NikkiChemical Co., Ltd., Yokohama, Japan, stirred at 100° C. for four hourswhile keeping the inner hydrogen pressure at 100 kg/cm², and stirred at120° C. for another two hours to effect hydrogenation. After standing tocool, the hydrogenated products were collected from the autoclave andpassed through an activated charcoal layer about 1-cm thick to removethe Raney nickel catalyst. The filtrate was in a usual manner desalted,purified, and concentrated to give a concentration of about 73%. Theconcentrate was placed in a cylindrical plastic vessel, admixed with0.1% to the solids, d.s.b., of a crystalline isomaltitol powder as aseed, cooled to 35° C. over about 20 hours under gentle stirringconditions to crystallize isomaltitol. Then, the resulting mixture wasseparated by a centrifuge to collect isomaltitol crystal, and dried invacuo at 80° C. for 20 hours to obtain about 168 g of isomaltitolcrystal.

The product had an isomaltitol purity of about 99.9% or higher, d.s.b.The results on x-ray powder diffraction pattern, ¹H-NMR spectrum, and¹³C-NMR spectrum of the product are respectively shown in FIGS. 48 to50. Based on the data, the product was judged to be isomaltitol.

The following Example A explains isomaltose or saccharides comprisingthe same and the process for producing isomaltitol and/or saccharidescomprising the same; and Example B explains the uses of isomaltitoland/or saccharides comprising the same:

EXAMPLE A-1

About one hundred liter of an aqueous solution of phytoglycogen fromcorn commercialized by Q.P. Corporation, Tokyo, Japan, was adjusted togive a concentration of 4% (w/v) and pH 6.0, heated to 30° C., andadmixed with one unit/g starch of a purified specimen ofα-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporusN75 strain obtained by the method in Experiment 11-2, and 12 units/gstarch of a purified specimen of α-isomaltosyl-transferring enzyme fromBacillus globisporus N75 strain obtained by the method in Experiment11-3, followed by an enzymatic reaction for 48 hours and a heattreatment at 100° C. for 10 min to inactivate the remaining enzymes. Themixture thus obtained was sampled for quantifying the yield ofcyclotetrasaccharide on HPLC, revealing that it had about 80% ofcyclotetrasaccharide in terms of sugar composition, where HPLC wascarried out using “SHOWDEX™ KS-801 column”, Showa Denko K.K., Tokyo,Japan, at a column temperature of 60° C. and a flow rate of 0.5 ml/minof water, and “RI-8012”, a differential refractometer commercialized byTosoh Corporation, Tokyo, Japan. The above mixture was adjusted to pH5.0 and 45° C., admixed with 1,500 units/g starch, d.s.b. of“TRANSGLUCOSIDASE L AMANO™”, an α-glucosidase commercialized by AmanoPharmaceutical Co., Ltd., Aichi, Japan, and 75 units/g starch, d.s.b.,of “XL-4™”, a glucoamylase specimen commercialized by NagaseBiochemicals, Ltd., Kyoto, Japan, incubated for 24 hours to hydrolyzethe remaining reducing oligosaccharides, etc. The resulting mixture wasadjusted to pH 5.8, kept at 90° C. for one hour to inactivate theremaining enzymes, and filtered to remove insoluble substances. Thefiltrate was concentrated to give a concentration of about 16% with“HOLLOSEP® HR 5155PI”, a reverse osmotic membrane, Toyobo Co., Ltd.,Tokyo, Japan, and in a usual manner decolored, desalted, filtered, andconcentrated to obtain about 6.0 kg of a saccharide solution with asolid content of about 3,500 g, d.s.b. The saccharide solution was fedto a column packed with about 225 L of “AMBERLITE CR-1310 (Na⁺-form)”, astrong-acid cation exchange resin commercialized by Japan Organo Co.,Ltd., Tokyo, Japan, and chromatographed at a column temperature of 60°C. and a flow rate of about 45 L/h. While the saccharide composition ofeluate from the column was monitoring by the above-identified HPLC,fractions of cyclotetrasaccharide with a purity of at least 80% werecollected, and in a usual manner desalted, decolored, filtered, andconcentrated into a saccharide solution.

HPLC analysis for saccharide composition of the saccharide solution thusobtained revealed that it contained cyclotetrasaccharide with a purityof about 95.5%. The resulting saccharide solution withcyclotetrasaccharide was concentrated in vacuo into a powder containingcyclotetrasaccharide. The powder was dissolved in deionized water,adjusted to give a concentration of one percent, pH 5.5 and 50° C., andadmixed with 80 units/g solids, d.s.b., of an isomaltose-releasingenzyme obtained by the method in Experiment 29, followed by an enzymaticreaction at pH 5.5 and 50° C. for 70 hours. Thereafter, the resultingmixture was sequentially heated to 95° C., kept at the temperature for10 min, cooled, and filtered. The filtrate was in a usual mannerdecolored with an activated charcoal, desalted for purification withion-exchange resins in H- and OH-forms, and concentrated to give aconcentration of about 43.0%. Thus, a high isomaltose content syrup wasobtained in a yield of about 95%, d.s.b., to the solid contents. HPLCanalysis for saccharide composition of the syrup revealed thus obtainedthat it contained 43.1% of isomaltose, 37.8% of ring-openedtetrasaccharide, and 13.8% of cyclotetrasaccharide.

The product has a satisfactory moisture-retaining ability, lowsweetness, osmosis-controlling ability, filler-imparting ability,gloss-imparting ability, viscosity-imparting ability, ability ofpreventing crystallization of other saccharides, insubstantialfermentability, ability of preventing the retrogradation of starch,etc., it can be arbitrarily used in various food products, health foods,feeds, pet foods including bait for fish, cosmetics, pharmaceuticals,and favorite foods.

EXAMPLE A-2

A powder containing cyclotetrasaccharide, obtained by the method inExample A-1, was dissolved in deionized water, adjusted to give aconcentration of one percent, pH 5.5 and 50° C., and admixed with 500units/g solids, d.s.b., of an isomaltose-releasing enzyme obtained bythe method in Experiment 29, followed by an enzymatic reaction at pH 5.5and 50° C. for 70 hours. Thereafter, the resulting mixture wassequentially heated to 95° C., kept at the temperature for 10 min,cooled, and filtered. The filtrate was in a usual manner decolored withan activated charcoal, desalted for purification with ion-exchangeresins in H- and OH-forms, and concentrated to give a concentration ofabout 75%. Thus, a high isomaltose content syrup was obtained in a yieldof about 90%, d.s.b., to the solid contents. HPLC analysis forsaccharide composition of the syrup revealed that it contained 92.8% ofisomaltose, 2.7% of ring-opened tetrasaccharide, and 4.5% of othersaccharides.

The product has a satisfactory moisture-retaining ability, lowsweetness, osmosis-controlling ability, filler-imparting ability,gloss-imparting ability, viscosity-imparting ability, ability ofpreventing crystallization of other saccharides, insubstantialfermentability, ability of preventing the retrogradation of starch,etc., it can be arbitrarily used in various food products, health foods,feeds, pet foods including bait for fish, cosmetics, pharmaceuticals,and favorite foods.

EXAMPLE A-3

An about 20% corn starch suspension was prepared, admixed with 0.1%calcium carbonate, adjusted to pH 6.5, and then mixed with 0.3% per gramstarch of “TERMAMYL 60L™”, an α-amylase specimen commercialized by NovoIndutri A/S, Copenhagen, Denmark, followed by an enzymatic reaction at95° C. for 15 min. Thereafter, the reaction mixture was autoclaved at120° C. for 20 min, and promptly cooled to about 50° C. to obtain aliquefied starch solution with a DE of about four. To the liquefiedsolution were added 0.2 unit/g solid, d.s.b., of a purified specimen ofα-isomaltosylglucosaccharide-forming enzyme from N75 strain obtained bythe method in Experiment 11-2, 100 units/g solid, d.s.b., of anisomaltodextranase specimen obtained by the method in Experiment 29, 250units/g solid, d.s.b., of an isoamylase specimen from Pseudomonasamyloderamosa commercialized by Hayashibara Biochemical laboratories,Inc., Okayama, Japan, and 0.5 unit/g starch of a CGTase specimen fromBacillus stearothermophilus commercialized by Hayashibara Biochemicallaboratories, Inc., Okayama, Japan, followed by an incubation at 50° C.and pH 5.5 for 65 hours. After completion of the reaction, the reactionmixture was heated at 100° C. for 15 min to inactivate the remainingenzymes. To the resulting mixture was added 20 units/g starch of“XL-4™”, a glucoamylase specimen commercialized by Nagase Biochemicals,Ltd., Kyoto, Japan, incubated at 50° C. for 24, and heated at 100° C.for 20 min to inactivate the remaining enzyme. The reaction mixture wascooled and filtered. The filtrate was in a usual manner decolored withan activated charcoal, desalted for purification with ion-exchangeresins in H- and OH-forms, and concentrated to give a concentration ofabout 60%. Thus, a high isomaltose content syrup was obtained in a yieldof about 95%, d.s.b., to the solid contents. HPLC analysis forsaccharide composition of the syrup revealed that it contained 62.9% ofisomaltose, 30.1% of glucose, and 7.0% of other saccharides.

The product has a satisfactory moisture-retaining ability, lowsweetness, osmosis-controlling ability, filler-imparting ability,gloss-imparting ability, viscosity-imparting ability, ability ofpreventing crystallization of other saccharides, insubstantialfermentability, ability of preventing the retrogradation of starch,etc., it can be arbitrarily used in various food products, health foods,feeds, pet foods including bait for fish, cosmetics, pharmaceuticals,and favorite foods.

EXAMPLE A-4

A high isomaltose content syrup, obtained by the method in Example A-3,as a saccharide solution, was column chromatographed to increase theconcentration of isomaltose using “AMBERLITE CR-1310 (Na⁺-form)”, astrong-acid cation exchange resin commercialized by Japan Organo Co.,Ltd., Tokyo, Japan, in such a manner of packing the above resin to 10stainless-steel columns equipped with an inner jacket having 12.5 cm indiameter, cascading the columns in series to give a total column beddepth of 16 m, applying the above syrup in a volume of 1.5% (v/v) to thevolume of resin, fractionating and purifying the syrup by feeding hotwater heated to 40° C. to the columns at a space velocity (SV) of 0.2,collecting fractions rich in isomaltose while monitoring the sugarcomposition of the eluates, and concentrating the pooled eluates up togive a concentration of 75% to obtain a high isomaltose content syrup,consisting of, on a dry solid basis, 4.3% glucose, 90.5% isomaltose,3.5% of other saccharides, and 1.7% of trisaccharide or higher, in ayield of about 45%.

The product has a satisfactory moisture-retaining ability, lowsweetness, osmosis-controlling ability, filler-imparting ability,gloss-imparting ability, viscosity-imparting ability, ability ofpreventing crystallization of other saccharides, insubstantialfermentability, ability of preventing the retrogradation of starch,etc., it can be arbitrarily used in various food products, health foods,feeds, pet foods including bait for fish, cosmetics, pharmaceuticals,and favorite foods.

EXAMPLE A-5

An isomaltose content syrup, obtained by the method in Example A-1, washydrogenated in accordance with the method in Experiment 36, and theresulting mixture was in a usual manner decolored with an activatedcharcoal, desalted for purification with ion-exchange resins in H- andOH-forms, and concentrated to give a concentration of about 73%. Theconcentrate was spray dried in a usual manner to obtain a highisomaltitol content powder, containing 43.3% of isomaltitol, 37.8% ofring-opened tetrasaccharide, 13.8% of cyclotetrasaccharide, and 3.5% ofother sugar alcohols, in a yield of about 80%.

The product is substantially a non-reducing saccharide which does notsubstantially cause the Maillard reaction and substantially hasnon-hygroscopicity, low sweetness, osmosis-controlling ability,filler-imparting ability, gloss-imparting ability, moisture-retainingability, viscosity-imparting ability, ability of preventingcrystallization of other saccharides, insubstantial fermentability,ability of preventing the retrogradation of starch, etc., it can bearbitrarily used in various food products, health foods, healthsupplements, feeds, pet foods including bait for fish, cosmetics,pharmaceuticals, and favorite foods.

EXAMPLE A-6

A 20% tapioca starch suspension was prepared, admixed with 0.1% calciumcarbonate, adjusted to pH 6.5, and then mixed with 0.3% per gram starchof “TERMAMYL 60L™”, an α-amylase specimen commercialized by Novo IndutriA/S, Copenhagen, Denmark, followed by an enzymatic reaction at 95° C.for 15 min. Thereafter, the reaction mixture was autoclaved at 120° C.for 20 min, and promptly cooled to about 40° C. to obtain a liquefiedstarch solution with a DE of about four. To the liquefied starchsolution were added 0.2 unit/g solid, d.s.b., of a purified specimen ofα-isomaltosylglucosaccharide-forming enzyme from C9 strain obtained bythe method in Experiment 4-2, 100 units/g solid, d.s.b., of a purifiedspecimen of α-isomaltodextranase obtained by the method in Experiment29, 250 units/g of an isoamylase specimen commercialized by HayashibaraBiochemical Laboratories, Inc., Okayama, Japan, and 0.5 unit/g of aCGTase specimen commercialized by Hayashibara Biochemical Laboratories,Inc., Okayama, Japan, followed by an incubation at pH 5.5 and 40° C. for64 hours. After completion of the reaction, the reaction mixture wassequentially heated at 95° C. for 30 min, cooled to 50° C., admixed with10 units/g of “GLUCOZYME™”, a glucoamylase specimen commercialized byNagase Biochemicals, Ltd., Kyoto, Japan, subjected to an enzymaticreaction for 24 hours, heated to 95° C., incubated at 95° C. for 30 min,cooled, and filtered. The filtrate was in a usual manner decolored withan activated charcoal, desalted for purification with ion-exchangeresins in H- and OH-forms, and concentrated to give a concentration ofabout 50% (w/v). Thus, a high isomaltose content syrup, containing 11.0%of glucose, 66.5% of isomaltose, 2.4% of disaccharide other thanisomaltose, and 20.1% of trisaccharide or higher, was obtained in ayield of about 95%, d.s.b.

The high isomaltose content syrup thus obtained was hydrogenated inaccordance with the method in Experiment 36, followed by removing theRaney Nickel catalyst from the mixture. The resulting mixture wasdecolored with an activated charcoal, desalted for purification withion-exchange resins in H- and OH-forms, concentrated, and dried in vacuoto obtain a high isomaltitol content powder in a yield of about 85%.

The powder contained 12.3% of sorbitol, 66.7% of isomaltitol, and 21.0%of other sugar alcohols.

The product substantially does not have reducibility and does not causethe Maillard reaction, and it has a relatively low sweetness,osmosis-controlling ability, filler-imparting ability, gloss-impartingability, moisture-retaining ability, viscosity-imparting ability,ability of preventing crystallization of other saccharides,insubstantial fermentability, ability of preventing the retrogradationof starch, etc. Thus the product can be arbitrarily used in various foodproducts, health foods, feeds, pet foods including bait for fish,cosmetics, pharmaceuticals, and favorite foods.

EXAMPLE A-7

In accordance with the method in Experiment 1, Bacillus globisporus C9strain (FERM BP-7143) was cultured in a fermentor for 48 hours.Thereafter, the culture was membrane filtered to remove the cells tocollect about 18 L of a filtrate which was then concentrated with a UFmembrane to yield about one liter of an enzyme concentrate containing8.8 units/ml of α-isomaltosylglucosaccharide-forming enzyme and 26.7units/ml of α-isomaltosyl-transferring enzyme. While, an about 27% cornstarch suspension was prepared, admixed with 0.1% calcium carbonate,adjusted to pH 6.5, and then mixed with 0.3% per gram starch of“TERMAMYL 60L™”, an α-amylase specimen commercialized by Novo IndutriA/S, Copenhagen, Denmark, followed by an enzymatic reaction at 95° C.for 15 min. Thereafter, the reaction mixture was autoclaved at 120° C.for 20 min and promptly cooled to about 40° C. to obtain a liquefiedstarch solution with a DE of about four. To the liquefied starchsolution were added 0.25 ml of the above enzyme solution ofα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme, 100 units/g starch of anisomaltodextranase specimen obtained by the method in Experiment 29, 250units/g starch of an isoamylase specimen commercialized by HayashibaraBiochemical Laboratories, Inc., Okayama, Japan, and 0.5 unit/g starch ofa CGTase specimen commercialized by Hayashibara BiochemicalLaboratories, Inc., Okayama, Japan, followed by an incubation at pH 5.5and 40° C. for 70 hours. After completion of the reaction, the reactionmixture was sequentially heated to 95° C., incubated at 95° C. for 10min, adjusted to 50° C., admixed with 20 units/g starch of “GLUCOZYME™”,a glucoamylase specimen commercialized by Nagase Biochemicals, Ltd.,Kyoto, Japan, enzymatically reacted for 24, and heated to and incubatedat 95° C. for 30 min. The resulting mixture was cooled and filtered. Thefiltrate was in a usual manner decolored with an activated charcoal,desalted for purification with ion-exchange resins in H- and OH-forms,and concentrated to give a concentration of about 50%. Thus, a highisomaltose content syrup, containing 32.6% glucose, 59.4% of isomaltose,1.2% of disaccharide other than isomaltose, 6.8% of trisaccharide orhigher, was obtained in a yield of about 95%, d.s.b.

The high isomaltose content syrup thus obtained was hydrogenated inaccordance with the method in Experiment 36, followed by removing theRaney Nickel catalyst from the mixture in a usual manner. The resultingmixture was decolored with an activated charcoal, desalted forpurification with ion-exchange resins in H- and OH-forms, concentratedto give a concentration of about 50%. Thus, a high isomaltitol contentsyrup was obtained in a yield of about 85%, d.s.b.

The product contained 33.4% of sorbitol, 59.1% of isomaltitol, 6.4% ofsugar alcohols other than sorbitol and isomaltitol, and 1.1% ofcyclotetrasaccharide. The product substantially does not hasreducibility and does not cause the Maillard reaction, and it has arelatively low sweetness, osmosis-controlling ability, filler-impartingability, gloss-imparting ability, moisture-retaining ability,viscosity-imparting ability, ability of preventing crystallization ofother saccharides, insubstantial fermentability, ability of preventingthe retrogradation of starch, etc. Thus the product can be arbitrarilyused in various food products, health foods, feeds, pet foods includingbait for fish, cosmetics, pharmaceuticals, and favorite foods.

EXAMPLE A-8

A high isomaltose content syrup, obtained by the method in Example A-7,as a saccharide solution, was column chromatographed to increase thecontent of isomaltose using “AMBERLITE CR-1310 (Na⁺-form)”, astrong-acid cation exchange resin commercialized by Japan Organo Co.,Ltd., Tokyo, Japan, in such a manner of packing the above resin to 10stainless-steel columns equipped with an inner jacket having 12.5 cm indiameter, cascading the columns in series to give a total column beddepth of 16 m, applying the above syrup in a volume of 1.5% (v/v) to thevolume of resin, fractionating and purifying the syrup by feeding hotwater heated to 40° C. to the columns at SV 0.2, collecting fractionsrich in isomaltose while monitoring the sugar composition of theeluates, and concentrating the pooled eluates up to give a concentrationof 55% to obtain a high isomaltose content syrup, consisting of, on adry solid basis, 4.8% glucose, 88.0% isomaltose, 4.1% of othersaccharides, and 3.1% of trisaccharide or higher, in a yield of about55%.

The high isomaltose content syrup thus obtained was hydrogenated inaccordance with the method in Experiment 36, followed by removing theRaney Nickel catalyst from the mixture in a usual manner. The resultingmixture was decolored with an activated charcoal and desalted forpurification with ion-exchange resins in H- and OH-forms to obtain ahigh isomaltitol content syrup, consisting of, on a dry solid basis,4.9% sorbitol, 88.1% isomaltitol, and 7.0% of other sugar alcohols, in ayield of about 90%.

The high isomaltitol content syrup thus obtained was concentrated togive a concentration of about 73%, and the concentrate was placed in acrystallizer, admixed with a crystalline isomaltitol powder as a seed inan amount of 0.1%, d.s.b., to the solid contents, and allowed tocrystallize maltitol at 25° C. for about 20 hours. The mixture wasseparated by a centrifuge, followed by separately collecting theresulting isomaltitol crystal and syrup. The isomaltitol crystal thusobtained was dried in vacuo at 80° C. for 20 hours to obtain acrystalline maltitol powder in a yield of about 39%, d.s.b. Inaccordance with the above method, the above syrup was columnchromatographed using a strong-acid cation exchange resin, followedcollecting high isomaltitol content fractions with an isomaltitolcontent of about 88%, d.s.b. The fractions were pooled, purified,concentrated, crystallized, and separated to collect isomaltitol crystalwhich was then aged and dried in vacuo to obtain a crystallineisomaltitol powder in a yield of about 20%, d.s.b. By combining thepowder thus obtained and the previously obtained powder, a crystallineisomaltitol powder was obtained in a total yield of about 59%, d.s.b.

The product contained, on a dry solid basis, 0.7% sorbitol, 98.0%isomaltitol, and 1.3% sugar alcohol. The product has non-reducibility,non-hygroscopicity, low sweetness, osmosis-controlling ability,filler-imparting ability, gloss-imparting ability, moisture-retainingability, viscosity-imparting ability, ability of preventingcrystallization of other saccharides, insubstantial fermentability,ability of preventing the retrogradation of starch, etc., it can bearbitrarily used in various food products, health foods, healthsupplements, feeds, pet foods including bait for fish, cosmetics,pharmaceuticals, and favorite foods.

EXAMPLE A-9

About one hundred liter of an aqueous solution of phytoglycogen fromcorn commercialized by Q.P. Corporation, Tokyo, Japan, was adjusted togive a concentration of 4% (w/v) and pH 6.0, heated to 30° C., andadmixed with one unit/g starch of a purified specimen ofα-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporusN75 strain obtained by the method in Experiment 11-2, and 12 units/gstarch of a purified specimen of α-isomaltosyl-transferring enzyme fromBacillus globisporus N75 strain obtained by the method in Experiment11-3, followed by an enzymatic reaction for 48 hours and a heattreatment at 100° C. for 10 min to inactivate the remaining enzymes. Themixture thus obtained was sampled for quantifying the yield ofcyclotetrasaccharide on HPLC, revealing that it contained about 80% ofcyclotetrasaccharide in terms of sugar composition, where HPLC wascarried out using “SHOWDEX KS-80™ column”, Showa Denko K.K., Tokyo,Japan, at a column temperature of 60° C. and a flow rate of 0.5 ml/minof water, and “RI-8012™”, a differential refractometer commercialized byTosoh Corporation, Tokyo, Japan. The above mixture was adjusted to pH5.0 and 45° C., admixed with 1,500 units/g starch, d.s.b. of“TRANSGLUCOSIDASE L AMANO™”, an α-glucosidase commercialized by AmanoPharmaceutical Co., Ltd., Aichi, Japan, and 75 units/g starch, d.s.b.,of “XL-4™”, a glucoamylase specimen commercialized by NagaseBiochemicals, Ltd., Kyoto, Japan, incubated for 24 hours to hydrolyzethe remaining reducing oligosaccharides, etc. The resulting mixture wasadjusted to pH 5.8, kept at 90° C. for one hour to inactivate theremaining enzymes, and filtered to remove insoluble substances. Thefiltrate was concentrated to give a concentration of about 16% (w/v)with “HOLLOSEP® HR 5155PI”, a reverse osmotic membrane, Toyobo Co.,Ltd., Tokyo, Japan, and in a usual manner decolored, desalted, filtered,and concentrated into a saccharide solution. Then, the saccharidesolution was adjusted to give concentration of about one percent, pH5.5, and 50° C., admixed with 80 units/g solids of an isomaltodextranasespecimen prepared by the method in Experiment 29, and subjected to anenzymatic reaction at pH 5.5 and 50° C. for 70 hours. Thereafter, thereaction mixture was heated to and incubated at 95° C. for 10 min,cooled, and filtered. The filtrate was in a usual manner decolored withan activated charcoal, desalted for purification with ion-exchangeresins in H- and OH-forms, and concentrated to give a concentration ofabout 43% (w/v). Thus, an isomaltose content syrup was obtained in ayield of about 95%, d.s.b. HPLC analysis for saccharide composition ofthe syrup revealed that it contained 35.5% of isomaltose. The isomaltosecontent syrup thus obtained was hydrogenated in accordance with themethod in Experiment 36, followed by removing the Raney Nickel catalystfrom the mixture in a usual manner. The resulting mixture was decoloredwith an activated charcoal, desalted for purification with ion-exchangeresins in H- and OH-forms, and concentrated to give a concentration ofabout 40%. The resulting concentrate was column chromatographed using acolumn packed with about 225 L of “AMBERLITE CR-1310 (Na⁺-form)”, astrong-acid cation exchange resin commercialized by Japan Organo Co.,Ltd., Tokyo, Japan, at a column temperature of 60° C. and a flow rate ofabout 45 L/h, followed by collecting fractions containing isomaltitolwith a purity of at least 50% while monitoring the saccharidecomposition on the above-identified HPLC. The fractions were pooled, andin a usual manner desalted for purification with ion-exchange resins inH- and OH-forms, decolored, filtered, and concentrated to give aconcentration of about 50%, d.s.b. Thus a high isomaltitol contentsyrup, containing 65.3% of isomaltitol, 13.8% of reduced ring-openedcyclotetrasaccharide, 5.2% of cyclotetrasaccharide, and 15.7% of sugaralcohols such as sorbitol, was obtained in a yield of about 78%, d.s.b.

The product is substantially free of the Maillard reaction, and it has asatisfactory osmosis-controlling ability, filler-imparting ability,gloss-imparting ability, moisture-retaining ability, viscosity-impartingability, non-fermentability, ability of preventing the retrogradation ofstarch, etc. Thus the product can be arbitrarily used in various foodproducts, health foods, feeds, pet foods including bait for fish,cosmetics, pharmaceuticals, and favorite foods.

EXAMPLE A-10

An high isomaltitol content syrup, consisting of 4.9% sorbitol, 88.1% ofisomaltitol, and 7.0% of other sugar alcohols, was concentrated to givea concentration of about 88%. The concentrate was placed in acrystallizer, admixed with crystalline isomaltitol powder in an amountof two percent to the contents, d.s.b., heated to 50° C., incubated fortwo hours under gentle stirring conditions, transferred to a vat,allowed to stand at 20° C. for four days to crystallize and solidify thecontents. The resulting solid product was pulverized by a cutter anddried to obtain a crystalline isomaltitol powder in a yield of about90%.

The product has non-reducibility, non-hygroscopicity, low sweetness,osmosis-controlling ability, filler-imparting ability, gloss-impartingability, moisture-retaining ability, viscosity-imparting ability,ability of preventing crystallization of other saccharides,insubstantial fermentability, ability of preventing the retrogradationof starch, etc., it can be arbitrarily used in various food products,health foods, health supplements, feeds, pet foods including bait forfish, cosmetics, pharmaceuticals, and favorite foods.

EXAMPLE B-1

Sweetener.

To 0.8 part by weight of a crystalline isomaltitol powder, obtained bythe method in Example A-8, were added to homogeneity 0.2 part by weightof “TREHA®”, an α,α-trehalose product commercialized by HayashibaraShoji, Inc., Okayama, Japan, 0.01 part by weight of “αG SWEET™”, anα-glycosyl stevioside commercialized by Toyo Sugar Refining Co., Ltd.,Tokyo, Japan, and 0.01 part by weight of “ASPARTAME™” or L-aspartylphenylalanine methyl ester. The mixture was subjected to a granulator toobtain a granular sweetener. The product, which does not substantiallyhas hygroscopicity but has satisfactory moisture-retaining ability andlow sweetness, is a stable sweetener containing isomaltitol free fromcausing deterioration even when stored at ambient temperature.

EXAMPLE B-2

Hard Candy

To 100 parts by weight of a 55% sucrose solution were added 50 parts byweight of a high isomaltitol content syrup obtained by the method inExample A-7, and the mixture was concentrated by heating under a reducedpressure to give a moisture content of less than two percent. Theconcentrate was admixed with 0.6 part by weight of citric acid andadequate amounts of a lemon flavor and a color, followed by shaping theresulting mixture into a hard candy. The product, which is only lesscolored by the Maillard reaction and is satisfactory in biting property,flavor, and taste, is a stable, high quality hard candy free fromcausing crystallization of sucrose and having lesser hygroscopicity.

EXAMPLE B-3

Chewing Gum

Three parts by weight of a gum base were melted by heating to an extentto be softened and then admixed with two parts by weight of anhydrouscrystalline maltitol, two parts by weight of xylitol, two parts byweight of a high isomaltitol content syrup obtained by the method inExample A-7, and one part by weight of hydrous crystallineα,α-trehalose, monohydrate, and further mixed with adequate amounts of aflavor and a color. The mixture was in a usual manner kneaded by a rolland then shaped and packed to obtain a chewing gum. The product is arelatively low cariogenic, caloric chewing gum having a satisfactorytexture, flavor, and taste.

EXAMPLE B-4

Chocolate

Forty parts by weight of a cacao paste, 10 parts by weight of a cacaobutter, and 50 parts by weight of a crystalline isomaltitol obtained bythe method in Example A-8 were mixed, and the mixture was fed to arefiner to reduce the granular size and then placed in a conche andkneaded at 50° C. over two days and nights. During the processing, 0.5part by weight of lecithin was added to the kneaded mixture and welldispersed therein. Thereafter, the resulting mixture was adjusted to 31°C. with a thermo controller, and then poured into a mold just beforesolidification of the butter, deairated by a vibrator, and solidified bypassing through a cooling tunnel kept at 10° C. over 20 min. Thesolidified contents were removed from the mold and packed to obtain achocolate.

The product substantially has no hygroscopicity but has satisfactorycolor, gloss, and internal texture; smoothly melts in the mouth; and hasa high quality sweetness and a mild taste and flavor. The product can beuseful as a low caloric, cariogenic chocolate.

EXAMPLE B-5

Powdery Peptide

One part by weight of 40% of “HINUTE S™”, a peptide solution of ediblesoy beans commercialized by Fuji Oil Co., Ltd., Tokyo, Japan, was mixedwith two parts by weight of a high isomaltitol content syrup obtained bythe method in Example A-6, and the resultant mixture was placed in aplastic vat, dried in vacuo at 50° C., and pulverized to obtain apowdery peptide. The product, which is only less colored by the Maillardreaction, is useful as a material for low caloric confectionery and alsoas a material for controlling intestinal function, health food, andhardly assimilable dietary fiber for oral or tube fed liquid diets.

EXAMPLE B-6

Bath Salt

One part by weight of a peel juice of “yuzu” (a Chinese lemon) wasadmixed with 10 parts by weight of a crystalline isomaltitol powderobtained in accordance with the method in Example A-10, and 10 parts byweight of anhydrous crystalline cyclotetrasaccharide, followed bycrystallizing hydrous cyclotetrasaccharide crystal, penta- orhexa-hydrate, aging the crystal and pulverizing the aged crystal toobtain an isomaltitol and cyclotetrasaccharide powder with a yuzuextract.

To five parts by weight of the powder thus obtained were added 90 partsby weight of roast salt, two parts by weight of hydrous crystallineα,α-trehalose, one part by weight of silicic anhydride, and 0.5 part byweight of “αG HESPERIDIN™”, α-glucosyl hesperidin commercialized byHayashibara Shoji, Inc., Okayama, Japan, to obtain a bath salt.

The product is a high quality bath salt enriched with yuzu flavor andused by diluting in a bathtub with hot water by 100-10,000 folds, and itmoisturizes and smooths the skin and does not make you feel cold after abath.

EXAMPLE B-7

Cosmetic Cream

Two parts by weight of polyoxyethylene glycol monostearate, five partsby weight of glyceryl monostearate, self-emulsifying, two parts byweight of a high isomaltitol content syrup obtained by the method inExample A-7, one part by weight of “αG RUTIN™”, α-glucosyl rutincommercialized by Hayashibara Shoji, Inc., Okayama, Japan, one part byweight of liquid petrolatum, 10 parts by weight of glyceryltri-2-ethylhexanoate, and an adequate amount of an antiseptic weredissolved by heating in a usual manner. The resultant solution wasadmixed with two parts by weight of L-lactic acid, five parts by weightof 1,3-butylene glycol, and 66 parts by weight of refined water, and theresultant mixture was emulsified by a homogenizer and admixed with anadequate amount of a flavor while stirring to obtain a cosmetic cream.The product exhibits an antioxidant activity and has a relatively highstability, and these render it advantageously useful as a high qualitysunscreen, skin-refining agent, and skin-whitening agent.

EXAMPLE B-8

Toothpaste

A toothpaste was obtained by mixing 45 parts by weight of calciumsecondary phosphate, 1.5 parts by weight of sodium lauryl sulfate, 25parts by weight of glycerine, 0.5 part by weight of polyoxyethylenesorbitan laurate, 15 parts by weight of a high isomaltitol content syrupobtained by the method in Example A-2, 0.02 part by weight ofsaccharine, 0.05 part by weight of an antiseptic, and 13 parts by weightof water. The product has an improved after taste and satisfactoryfeeling after use without lowering the detergent power of thesurfactant.

EXAMPLE B-9

Solid Preparation for Fluid Diet

A composition was prepared by mixing 100 parts by weight of a highisomaltitol content powder obtained by the method in Example A-5, 200parts by weight of hydrous crystalline α,α-trehalose, 200 parts byweight of a high maltotetraose content powder, 270 parts by weight of anegg yolk powder, 209 parts by weight of a skim milk powder, 4.4 parts byweight of sodium chloride, 1.8 parts by weight of potassium chloride,four parts by weight of magnesium sulfate, 0.01 part by weight ofthiamine, 0.1 part by weight of sodium L-ascorbate, 0.6 part by weightof vitamin E acetate, and 0.04 part by weight of nicotinamide.Twenty-five gram aliquots of the composition were injected intomoisture-proof laminated small bags which were then heat-sealed toobtain the desired product.

The product is a fluid diet having a satisfactory action of improvingintestinal function. In use, one bag of the product is dissolved inabout 150 to about 300 ml of water into a fluid diet and arbitrarilyadministered orally or administered intubationally into the nasalcavity, stomach, intestines, etc.

EXAMPLE B-10

Tablet

Fifty parts by weight of aspirin were sufficiently mixed with 14 partsby weight of a crystalline isomaltitol powder obtained by the method inExample A-7, and four parts by weight of corn starch. The resultingmixture was in a usual manner tabletted by a tabletting machine toobtain a tablet, 680 mg and 5.25 mm in thickness.

The tablet, processed by using the filler-imparting ability ofisomaltitol, has substantially no hygroscopicity, but has a sufficientphysical strength and satisfactory degradability in water.

EXAMPLE B-11

Sugar Coated Tablet

A crude tablet as a core, 150 mg weight, was sugar coated with a firstsolution, consisting of 40 parts by weight of a crystalline isomaltitolobtained by the method in Experiment 36, two parts by weight of pullulanhaving an average molecular weight of 200,000, 30 parts by weight ofwater, 25 parts by weight of talc, and three parts by weight of titaniumoxide until the total weight increased to about 230 mg. The resultanttablet was further sugar coated with a second solution, consisting of 65parts by weight of a powder of hydrous crystalline cyclotetrasaccharide,penta- or hexa-hydrate, one part by weight of pullulan, and 34 parts byweight of water. Then, the resulting tablet was glossed with a liquidwax into a sugar coated tablet having a satisfactory gloss andappearance. The product has a relatively high shock tolerance andretains its initial high quality for a relatively-long period of time.

EXAMPLE B-12

Ointment for Treating Trauma

To 100 parts by weight of a high isomaltitol content syrup, obtained bythe method in Example A-7, and 300 parts by weight of maltose were added50 parts by weight of methanol dissolving three parts by weight ofiodine, and further added 200 parts by weight of a 10% (w/v) aqueouspullulan solution to obtain the desired product with an adequateextensibility and adhesiveness. The product is a high-valued ointment inwhich the volatilization of iodine and methanol is well inhibited byisomaltitol and is relatively less in property change during storage.

Because the product exerts a sterilizing action by iodine and acts as anenergy-supplementing agent on living cells due to maltose, it shortensthe curing term and well cures the affected parts and surfaces.

INDUSTRIAL APPLICABILITY

As described above, the present invention relates to a novel method forproducing isomaltose and isomaltitol, more particularly, to a processfor producing isomaltose, which comprises the steps of contacting asaccharide, having the α-1,4 glucosidic linkage as the linkage ofnon-reducing end and a glucose polymerization degree of at least two,with one or more α-isomaltosylglucosaccharide-forming enzymes derivedfrom Bacillus globisporus N75 strain (FERM BP-7591), Arthrobacterglobiformis A19 strain (FERM BP-7590) and Arthrobacter ramosus S1 strain(FERM BP-7592) in the presence or the absence of anα-isomaltosyl-transferring enzyme derived from Bacillus globisporus N75strain (FERM BP-7591) and/or Arthrobacter globiformis A19 strain (FERMBP-7590) to form α-isomaltosylglucosaccharides having the α-1,6glucosidic linkage as the linkage of non-reducing end and the α-1,4glucosidic linkage other than the above linkage, and/or to form asaccharide with the structure ofcyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→};contacting the resulting mixture with isomaltose-releasing enzyme toform isomaltose; and collecting the produced isomaltose. The presentinvention also relates to a method for producing isomaltitol, whichcomprises the steps of contacting a saccharide, having the α-1,4glucosidic linkage as the linkage of non-reducing end and a glucosepolymerization degree of at least two, withα-isomaltosylglucosaccharide-forming enzyme to formα-isomaltosylglucosaccharides having the α-1,6 glucosidic linkage as thelinkage of non-reducing end and the α-1,4 glucosidic linkage other thanthe above linkage, and/or to form a saccharide with the structure ofcyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→};contacting the resulting mixture with isomaltose-releasing enzyme toform isomaltose; hydrogenating either the resulting mixture directly orthe isomaltose separated from the mixture to form isomaltitol; andcollecting the formed isomaltitol. The present invention further relatesto saccharide compositions containing isomaltose and/or isomaltitol, anduses thereof. According to the present invention, saccharidecompositions containing isomaltose and/or isomaltitol, which are usefulin this art, can be produced on an industrial scale, at a relatively lowcost and in a relatively high yield. The saccharide compositions of thepresent invention can be arbitrarily used in various food products,health foods, health supplements, feeds, pet foods including bait forfish, cosmetics, pharmaceuticals, and favorite foods because thecompositions, which are substantially free of reducibility and theMaillard reaction, have satisfactory low sweetness, osmosis-controllingability, filler-imparting ability, gloss-imparting ability,moisture-retaining ability, viscosity-imparting ability, ability ofpreventing crystallization of other saccharides, insubstantialfermentability, ability of preventing the retrogradation of starch, etc.

The present invention with these outstanding functions and effects is asignificant invention that greatly contributes to this art.

1. A process for producing isomaltitol, comprising the steps of: (a)allowing an α-isomaltosylglucosaccharide-forming enzyme, which forms anα-isomaltosylglucosaccharide with a glucose polymerization degree of atleast three and having both the α-1,6 glucosidic linkage as the linkageat the non-reducing end and the α-1,4 glucosidic linkage other than theabove linkage, via the α-glucosyl-transfer from a material saccharidehaving a glucose polymerization degree of at least two and having theα-1,4 glucosidic linkage as the linkage at the non-reducing end, withoutsubstantially increasing the reducing power of the material saccharide,to act on a saccharide with a glucose polymerization degree of at leasttwo and having the α-1,4 glucosidic linkage as the linkage ofnon-reducing end to form said α-isomaltosylglucosaccharide wherein saidα-isomaltosylglucosaccharide-forming enzyme has the followingphysicochemical properties: (1) Molecular weight Having a molecularweight of about 117,000 to about 160,000 daltons when determined onSDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis);(2) Isoelectric point Having an isoelectic point of about 4.7 to about5.7 when determined on isoelectrophoresis using ampholine; (3) Optimumtemperature Having an optimum temperature of about 40° C. to about 45°C. when incubated at a pH of 6.0 for 60 min; Having an optimumtemperature of about 45° C. to about 50° C. when incubated at a pH of6.0 for 60 min in the presence of 1 mM Ca²⁺; (4) Optimum pH Havingoptimum pH of about 6.0 to about 6.5 when incubated at 35° C. or 60 min;(5) Thermal stability Being stable up to a temperature of about 35° C.to 40° C. when incubated at a pH of 6.0 for 60 min, Being stable up to atemperature of about 40° C. to 45° C. when incubated at a pH of 6.0 for60 min in the presence of 1 mM Ca²⁺, (6) pH Stability Having a stable pHrange at about 4.5 to about 10.0 when incubated at 4° C. for 24 hours;(b) allowing an isomaltodextranase to act on the resulting mixture inthe step (a) to form isomaltose; (c) hydrogenating either the resultingmixture in the step (b) directly or the isomaltose, which has beenseparated from the mixture to form isomaltitol; and (d) collecting theformed isomaltitol.
 2. The process of claim 1, wherein one or moreenzymes selected from the group consisting of α-isomaltosyl-transferringenzyme, which forms a cyclotetrasaccharide having the structure ofcyclo{→6) -α-D-glucopyranosyl- (1→3) -α-D-glucopyranosyl- (1→6)-α-D-glucopyranosyl- (1→3) -α-D-glucopyranosyl- (1→}from saidα-isomaltosylglucosaccharide and has the following physicochemicalproperties: (1) Molecular weight Having a molecular weight of about82,000 to about 136,000 daltons when determined on SDS-PAGE; (2)Isoelectic point (pI) Having a pI about 5.0 to about 6.1 when determinedon isoelectrophoresis using ampholine; (3) Optimum temperature Having anoptimum temperature of about 45° C. to about 50° C. when incubated at apH of 6.0 for 30 min; (4) Optimum pH Having an optimum pH of about 5.5to about 6.0 when incubated at 35° C. for 30 min; (5) Thermal stabilityBeing stable up to a temperature of about 40° C. when incubated at a pHof 6.0 for 60 min; and (6) pH Stability Having a stable pH range atabout 4.0 to about 9.0 when incubated at 4° C. for 24 hours;cyclomaltodextrin glucanotransferase and starch debranching enzyme arefurther allowed to act on said saccharide with a glucose polymerizationdegree of at least two and having the α-1,4 glucosidic linkage as thelinkage of non-reducing end in the step (a).
 3. The process of claim 1,wherein glucoamylases is further allowed to act on the reaction mixtureafter the enzymatic reaction of said isomaltodextranase in the step (b).4. A process of claim 1, wherein said saccharide, having the α-1,4glucosidic linkage as the linkage of non-reducing end and a glucosepolymerization degree of at least two, is one or more saccharidesselected from the group consisting of maltooligosaccharides,maltodextrins, amylodextrins, amyloses, amylopectins, soluble starches,liquefied starches, gelatinized starches, and glycogens.
 5. The processof claim 1, characterized in that it employs a column chromatographyusing an alkaline metal- and/or alkaline earthmetal-strong-acid-cation-exchange-resin and optionally employs a step ofpulverization or crystallization in the step (d).
 6. The process ofclaim 1, wherein said isomaltitol is collected in the form of a syrup,powder, or crystal in the step (d).
 7. The process of claim 1, whereinthe collected isomaltitol in the step (d) is a high isomaltitol contentsyrup comprising isomaltitol in an amount of at least 40% (w/w), on adry solid basis.